Prostaglandin receptor protein

ABSTRACT

Described herein is a novel member of the prostanoid receptor family, a guinea pig prostaglandin D2 receptor. Described are the receptor, the nucleic acid that encodes it, and various uses for both.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/747,994 filed on Dec. 30, 2003, now U.S. Pat. No. 7,183,062issued Feb. 27, 2007, the disclosure of which is incorporated herein inits entirety.

FIELD OF THE INVENTION

The present invention relates generally to a nucleic acid molecule thatencodes a heretofore unknown member of the prostanoid receptor family.

BACKGROUND OF THE INVENTION

Prostanoids, including prostaglandin (PG), prostacyclin and thromboxane(TX), are important mediators of central and peripheral physiologicaleffects. Prostaglandin D2 (PGD2) is formed in a variety of tissuesincluding brain, spleen, lung, bone marrow, stomach, skin and also inmast cells (Lewis et al., 1982). PGD2 has been implicated in manyphysiological events both in the central nervous system and in theperipheral tissues. In the central nervous system, PGD2 has been shownto affect the induction of sleep, body temperature, olfactory function,hormone release and nociception. Peripherally, PGD2 is the majorcyclooxygenase product of arachidonic acid produced from mast cellsfollowing immunological challenge. Local allergen challenge in patientswith allergic rhinitis, bronchial asthma, allergic conjunctivitis andatopic dermatitis has been shown to result in rapid elevation of PGD2levels in nasal and bronchial lavage fluids, tears and skin chamberfluids. Activated mast cells, a major source of PGD2, are one of the keyplayers in driving the allergic response in conditions such as asthma,allergic rhinitis, allergic conjunctivitis, allergic dermatitis andother diseases (Brightling et al., 2003). Likewise, PGD2 has manyinflammatory actions, such as increasing vascular permeability in theconjunctiva and skin, increasing nasal airway resistance, airwaynarrowing and eosinophil infiltration into the conjunctiva and trachea.Therefore, PGD2 is considered one of the key players in drivinginflammatory reactions.

Early efforts have focused on identifying distinct receptors for thefive naturally occurring bioactive prostanoids, PGD₂, PGE₂, PGF_(2α),PGI₂ and TXA₂, resulting in the classification of five basic types ofprostanoid receptors: DP, EP, FP, prostacyclin (IP) and thomboxane (TP)receptors, respectively (Coleman et al., 1994). Many of the actions ofprostaglandin D2 are mediated through its action on the D-typeprostaglandin receptor (DP), a G protein-coupled receptor. Whileoriginally thought that each prostanoid acted preferentially onindividual receptors, researchers studying prostanoid biology have begunto appreciate the promiscuity of these ligands to interact with membersof the different receptor families. Thus, it is becoming ever more clearthat to understand prostanoid signaling one must elucidate thebiological consequence of prostanoid receptor activation.

The DP receptor is of particular interest because it is found in bothcentral and peripheral cells suggesting its involvement in mediatingvaried biological pathways and, consequently, its potential therapeuticimportance in many disease states. DP receptors have been identified inthe brain and PGD₂ has effects on sleep induction, body temperature,olfactory function, and hormone release (Negishi, et al., 1993; Wrightet al., 1999 and references within). DP receptors have also beenlocalized to discrete and distinct cell populations of the spinal cord.This observation may explain the discordant effects of hyperalgesia andallodynia (discomfort from innocuous tactile stimuli) induced by PGD₂.DP receptors are also present in the gastrointestinal tract and havebeen implicated in the contractile response of the GI tract (Wright etal., 1999; Ito et al., 1989). Additionally, DP receptor ligands havebeen shown to induce mucous secretion and cell proliferation ofintestinal cells. Glycogenesis in the liver may also be regulated by DPreceptors (Ito et al., 1989). DP receptors are found in the eye andagonists reduce intraoccular pressure suggesting a role in glaucoma.Platelets contain the DP receptor and PGD₂ has been shown to inhibitplatelet aggregation supporting a role for the DP receptor in modulatingblood disorders such as thrombosis (Armstrong, 1996). Thus, the variedexpression of the DP receptor in different organs and tissues suggeststhe DP receptor may be an attractive target for different therapeuticareas.

Of particular interest, the DP receptor has been implicated in variousinflammatory disorders including but not limited to asthma, allergicrhinitis, airway hyperactivity, allergic dermatitis, allergicconjunctivitis and chronic obstructive pulmonary disease. This issupported by the observation that PGD₂ is the major prostanoid releasedby immunochallenged mast cells (Roberts, et al., 1980). In asthma, therespiratory epithelium has long been recognized as a key source ofinflammatory cytokines and chemokines that drive the progression of thedisease (Holgate et al., 2000). In an experimental murine model ofasthma, the DP receptor is dramatically upregulated on airway epitheliumon antigen challenge (Matsuoka et al., 2000). Conversely, in knockoutmice lacking the DP receptor, there is a marked reduction in airwayhyperreactivity and chronic inflammation (Matsuoka et al., 2000); two ofthe cardinal features of human asthma. Similarly, DP receptorantagonists have been shown to reduce airway inflammation in a guineapig experimental asthma model (Arimura et al., 2001). The DP receptor isalso thought to be involved in human allergic rhinitis, a frequentallergic disease that is characterized by the symptoms of sneezing,itching, rhinorea and nasal congestion. Local administration of PGD₂ tothe nose causes a dose dependent increase in nasal congestion (Doyle etal. 1990). DP antagonists have been shown to be effective at alleviatingthe symptoms of allergic rhinitis in multiple species, and morespecifically, have been shown to inhibit the antigen-induced nasalcongestion, the most manifest symptom of allergic rhinitis. DPantagonists are also effective in experimental models of allergicconjunctivitis and allergic dermatitis (Arimura et al., 2001). Thus, DPantagonists could therefore be useful in the treatment of a variety ofPGD2-mediated disorders including, but not limited to, bronchial asthma,Chronic obstructive pulmonary disease (COPD), allergic rhinitis,allergic dermatitis, allergic conjunctivitis, systemic mastocytosis andischemic repurfusion injury.

Thus far, the DP receptor has been cloned from human (Boie et al.,1995), rat (Wright et al., 1999) and mouse (Hirata et al., 1994). TheseDP receptors share 73-90% homology at the amino acid level betweenhuman, mouse and rat and, in each case, activation of the recombinantreceptors leads to accumulation of intracellular cAMP. It is generallyobserved between G protein coupled receptors that compounds often showvarying potencies from one orthologue receptor to another.

Disclosed here, for the first time, is a DP receptor from the guineapig. The present invention provides several advantages over that whichis currently known in the art. Species differences between mouse, rat,human and guinea pig can now be more fully determined and characterized.The low expression levels of the DP receptor in native tissues makes itdifficult to assess a compound's activity as a modulator, effector,agonist or antagonist of the receptor. The present invention nowprovides the opportunity to examine the receptor in an isolated andpurified condition providing the ability to test compounds and then tobridge in vitro studies to the same species in vivo. Because of itslarger size, the guinea pig is a preferred animal model to smallerrodents, for instance, providing more surface area with regard todermatology and gastrointestinal studies. More importantly, the guineapig is the most usable small animal model for some allergy models suchas nasal congestion and is more responsive to airway hyperactivitymanipulations. Although the guinea pig represents an ideal preclinicalmodel for the evaluation of DP receptor modulators in multiple diseasemodels, as outlined above, the cloning of the guinea DP receptor has notpreviously been reported and hence it is difficult to predict theaffinity of a compound against this orthologue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleic acid sequence of the receptor of the invention(SEQ ID NO:1).

FIG. 2 shows an amino acid sequence of the receptor of the invention(SEQ ID NO:2).

FIG. 3 shows an alignment of the coding sequences of the DP receptorbetween multiple species. Shaded residues match the guinea pig residuesexactly. Hs=human (SEQ ID NO: 12); Rn=rat (SEQ ID NO: 13); Mm=mouse (SEQID NO: 14); and Cp=guinea pig (SEQ ID NO: 1).

FIG. 4 shows an alignment of the amino acid sequences of the DP receptorbetween multiple species. Shaded residues match the guinea pig residuesexactly. Hs=human (SEQ ID NO: 15); Rn=rat (SEQ ID NO: 16); Mm=mouse (SEQID NO: 17); Cp=guinea pig (SEQ ID NO: 2); Majority (SEQ ID NO: 18).

FIG. 5 shows a Northern blot analysis of genomic DNA fragment of theCavia porcellus DP receptor. Lane 1: Invitrogen 0.24-9.5 Kb RNA ladder;Lane 2: Total lung RNA from Cavia porcellus unchallenged; Lane 3: Totallung RNA from Cavia porcellus ovalbumin challenged.

FIG. 6 shows an example of PGD2-induced calcium mobilization ofrecombinant guinea pig DP receptor (SEQ ID NO: 2) expressed inHEK-293-Gα16 cells stably transfected with SEQ ID NO: 1 compared to anequivalent cell line generated with the mouse DP receptor and theparental cell line.

FIG. 7 shows an example PGD2 dose response curve of recombinant guineapig DP receptor (SEQ ID NO: 2) expressed in HEK-293-Ga16 cells stablytransfected with SEQ ID NO: 1 using the SPA cAMP assay. Comparison to anequivalent cell line generated with the mouse DP receptor and theparental cell line are included.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated nucleic acid and protein formsthat represent but are not necessarily limited to the prostanoidreceptor family. In a preferred embodiment, the isolated nucleic acidand protein represents the guinea pig DP receptor. Various aspects ofthe invention are described in further detail in the followingsubsections.

Definitions

As used herein, “nucleic acid molecule” refers to the phosphate esterpolymeric form of ribonucleosides (adenosine, guanosine, uridine orcytidine; “RNA”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA”) or anyphosphorester analogs thereof, such as phosphorothioates and thioesters,in either single stranded form, or double stranded helix form. Thenucleic acid molecule may include a deoxyribonucleic acid molecule(DNA), such as genomic DNA and complementary DNA (cDNA) that may becoding or noncoding single-stranded or double stranded, synthetic DNA,ribonucleic acid (RNA) molecule that may be single-stranded ordouble-stranded and analogs of the DNA and RNA generated usingnucleotide analogs. Double stranded DNA:DNA, DNA:RNA and RNA:RNA helicesare possible.

As used herein, an “isolated” or “purified” nucleic acid molecule is onethat is separated from other nucleic acid molecules that are present inthe natural source of the nucleic acid. Preferably, the “isolated”nucleic acid is free of sequences (preferably protein encodingsequences) which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. In variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences that flank the nucleotide molecule of the present invention.For example, these flanking nucleotide sequences may be sequences thatnaturally flank the nucleotide molecule in genomic DNA of the cell fromwhich the nucleic acid was isolated. A nucleic acid may be consideredisolated when it has been substantially removed from its endogenousenvironment to enable manipulation by one skilled in the art, such asbut not limited to nucleotide sequencing, restriction digestion,site-directed mutagenesis, and subcloning into expression vectors. Thenucleic acid may be present in whole cells or cell lysates or inpartially purified or substantially purified form. A nucleic acidpurified from cells is substantially free of other cellular material orculture medium. A chemically synthesized nucleic acid is purified whenit is substantially free of chemical precursors or other chemicals.

The term “recombinant,” when used in connection with a polypeptide,refers to a polypeptide derived from the translation of a recombinantpolynucleotide, that is, a polynucleotide that is isolated or purified(as defined above) or that is otherwise not in its native state. Theterm includes, for example, those polypeptides that are expressed by orcontained within cells that contain either a cloning vector orexpression vector, as well as synthetic polypeptides.

As used herein, the term “modulator” refers to a moiety (e.g., but notlimited to, a ligand and a candidate compound) that modulates theactivity of the receptor protein of the present invention. A modulatorof the present invention may be an agonist, a partial agonist, anantagonist, or an inverse agonist.

As used herein, the term “agonist” refers to moieties (e.g., but notlimited to, ligands and candidate compounds) that activate theintracellular response when bound to the receptor, or enhance GTPbinding to membranes.

As used herein, the term “partial agonist” refers to moieties (e.g., butnot limited to, ligands and candidate compounds) that activate theintracellular response when bound to the receptor to a lesserdegree/extent than do agonists, or enhance GTP binding to membranes to alesser degree/extent than do agonists.

As used herein, the term “antagonist” refers to moieties (e.g., but notlimited to, ligands and candidate compounds) that competitively bind tothe receptor at the same site as does an agonist. However, an antagonistdoes not activate the intracellular response initiated by the activeform of the receptor and thereby can inhibit the intracellular responsesby agonists or partial agonists. In a related aspect, antagonists do notdiminish the baseline intracellular response in the absence of anagonist or partial agonist.

As used herein, the term “inverse agonist” refers to moieties (e.g., butnot limited to, ligand and candidate compound) that bind to aconstitutively active receptor and inhibit the baseline intracellularresponse. The baseline response is initiated by the active form of thereceptor below the normal base level of activity that is observed in theabsence of agonists or partial agonists, or decrease of GTP binding tomembranes.

As used herein, the term “candidate compound” refers to a moiety (e.g.,but not limited to, a chemical compound) that is amenable to a screeningtechnique. In one embodiment, the term does not include compounds thatwere publicly known to be compounds selected from the group consistingof agonist, partial agonist, inverse agonist or antagonist. Thosecompounds were identified by traditional drug discovery processesinvolving identification of an endogenous ligand specific for areceptor, and/or screening of candidate compounds against a receptorwherein such a screening requires a competitive assay to assessefficacy.

As used herein, the terms “constitutively activated receptor” or“autonomously active receptor,” are used herein interchangeably, andrefer to a receptor subject to activation in the absence of ligand. Suchconstitutively active receptors can be endogenous or non-endogenous(i.e., GPCRs can be modified by recombinant means to produce mutantconstitutive forms of wild-type GPCRs; e.g., see EP 1071701; WO00/22129; WO 00/22131; and U.S. Pat. Nos. 6,150,393 and 6,140,509 whichare hereby incorporated by reference in their entireties).

As used herein, the term “constitutive receptor activation” refers tothe stabilization of a receptor in the active state by means other thanbinding of the receptor with the endogenous ligand or chemicalequivalent thereof.

As used herein, the term “ligand” refers to a moiety that binds toanother molecule, wherein the moiety includes, but certainly is notlimited to a hormone or a neurotransmitter, and further, wherein themoiety stereoselectively binds to a receptor.

As used herein, the term “family,” when referring to a protein or anucleic acid molecule of the invention, is intended to mean two or moreproteins or nucleic acid molecules having a seemingly common structuraldomain and having sufficient amino acid or nucleotide sequence identityas defined herein. Such family members can be naturally occurring andcan be from either the same or different species. For example, a familycan contain a first protein of human origin and a homologue of thatprotein of murine origin, as well as a second, distinct protein of humanorigin and a murine homologue of that second protein. Members of afamily also may have common functional characteristics.

As used herein interchangeably, the terms “activity”, “biologicalactivity” and “functional activity”, refer to an activity exerted by aprotein, polypeptide or nucleic acid molecule of the present inventionon a responsive cell as determined in vivo or in vitro, according tostandard techniques. An activity can be a direct activity, such as anassociation with or an enzymatic activity on a second protein or anindirect activity, such as a cellular signaling activity mediated byinteraction of the protein of the present invention with a secondprotein. In a particular embodiment, an activity includes, but is notlimited to at least one or more of the following activities: (i) theability to interact with proteins in the signaling pathway; (ii) theability to interact with a ligand; and (iii) the ability to interactwith an intracellular target protein.

Furthermore, in accordance with the present invention there may beemployed conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

A “vector” is a replicon, such as plasmid, phage or cosmid, to name onlya few, to which another DNA segment may be attached so as to bring aboutthe replication of the attached segment. A “replicon” is any geneticelement (e.g., plasmid, chromosome, virus) that functions as anautonomous unit of DNA replication in vivo, i.e., capable of replicationunder its own control. Particular examples of vectors are describedinfra.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. In particular, the vector targetsa specific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

Isolated Nucleic Acid Molecules

An aspect of the invention relates to isolated or purified nucleic acidmolecules that encode the receptor proteins of the invention or portionsthereof. The nucleic acid molecule of the present invention or acomplement of the nucleic acid sequence can be isolated using standardmolecular biology techniques and the sequence information provided inthe present invention. Using all or a portion of the nucleic acidsequence of SEQ NO:1 as a hybridization probe, nucleic acid molecules ofthe invention can be isolated using standard hybridization and cloningtechniques (Sambrook et al., 1989). Oligonucleotides corresponding toSEQ ID NO:1, or a portion thereof, can be prepared by standard synthetictechniques, e.g., using an automated DNA synthesizer. The nucleic acidmolecule of the invention, or part thereof, can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques.

The nucleic acid molecule of the invention can comprise a portion of SEQID NO:1. The nucleic acid fragment can be used as a probe or primer orthe fragment can encode a protein fragment that may or may not be abiologically active portion of the receptor such as the ligand bindingdomain. For instance, the arginine in the seventh transmembrane domainwas proposed to be the binding site for the carboxyl group of theprostanoid molecule (Narumiya et al., 1993) and Lys-75 and Leu-83 of thesecond transmembrane domain in the mouse confers ligand bindingspecificity (Kobayashi et al., 2000). These two sequence stretches havepreviously been reported to be characteristically conserved amongstGPCRs of the prostanoid family (Hirata et al., 1994) and are alsopresent in the guinea pig DP protein: QYCPGTWCR (SEQ ID NO: 10) in thesecond extracellular loop and RFLSVISIVDPWIFI (SEQ ID NO: 11) in theseventh transmembrane domain were identical among all DP orthologues.The nucleotide sequence of SEQ ID NO:1 allows for the generation ofprobes and primers for the use of identifying and/or cloning thereceptor of the invention or homologues in cells, tissues and organs.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least 10, preferablyabout 12, more preferably 25, 50, 75, 100, 125, 150, 175, 200, 250, 300,350 or 400 consecutive nucleotides of the sense or antisense sequence ofSEQ ID NO:1 or of a naturally occurring or man-made mutation of SEQ IDNO:1. The probe may comprise a label group attached thereto, e.g., aradioisotope, a fluorescent compound, an enzyme or an enzyme co-factor.The probe can be part of a kit for identifying cells or tissues encodingthe nucleic acid, detecting mRNA levels or determining whether a genomicgene has been mutated or deleted.

The present invention further extends to an isolated nucleic acidmolecule that is 90% homologous to SEQ ID NO:1. Sequences that aresubstantially homologous can be identified by comparing the sequencesusing standard software available in sequence data banks using defaultparameters, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

DNA sequence polymorphisms may exist within a population due to naturalallelic variation. An allele is one group of genes that occuralternatively at a given genetic locus. As used herein, the terms “gene”and “recombinant gene” refer to nucleic acid molecules comprising anopen reading frame encoding the receptor protein of the invention,preferably a guinea pig receptor protein. As used herein, the phrase“allelic variant” refers to a nucleotide sequence that occurs at thegene locus or to a polypeptide encoded by the nucleotide sequence.Alternative alleles can be identified by sequencing the gene of interestin a number of different individuals. Any and all such nucleotidevariations and resulting amino acid polymorphisms or variations that arethe result of natural allelic variation and that do not alter thefunctional activity of the receptor of the invention are intended to bewithin the scope of the invention.

A nucleic acid fragment encoding a “biologically active” or“biologically relevant” portion can be prepared by isolating a portionof SEQ ID NO:1 that encodes a polypeptide having the biological activityof the receptor of the invention. For instance, expressing the encodedportion of the receptor protein (e.g., by recombinant expression invitro) of the ligand-binding domain or the signal-transducing domain andthen assessing the activity of that encoded portion of the receptor. Theinvention further encompasses nucleic acid molecules that differ fromthe nucleotide sequence of SEQ ID NO:1 due to degeneracy of the geneticcode and thus encode same protein as that encoded by the nucleotidesequence of SEQ ID NO:1. For example, the inventors have identified twopotential N-glycosylation sites, Asn-7 in the amino terminus and Asn-86in the first extracellular loop. Additionally, there are also twopotential protein kinase C phosphorylation sites, Ser-46 and Thr-140located in the first and third cytoplasmic loops, respectively.

In addition to naturally occurring allelic variants, it is known bythose skilled in the art that there is substantial amount of redundancyin the various codons that code for specific amino acids. Thus, theinvention is directed to those DNA sequences encoding RNA comprisingalternative codons or RNA sequences encoding alternative codons whichcode for the eventual translation of the identical amino acid sequenceof SEQ ID NO:2 or portions thereof. It is well known in the art that thefollowing codons can be used interchangeably to code for each specificamino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA or UAG or UGA

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

A person skilled in the art can further appreciate that changes can beintroduced into SEQ ID NO:1 by mutation without altering the biologicalactivity of the encoded protein. A “non-essential” amino acid residue isa residue that can be altered from the wild-type sequence, e.g., thesequence of SEQ ID NO:2 without altering the biological activity whereasthe “essential” amino acid residues are required for biologicalactivity. Thus, amino acid residues that are not conserved or onlysemi-conserved among several species may be non-essential and likelytargets for alteration. Another aspect of the invention pertains tonucleic acid molecules encoding proteins of the invention that containchanges in amino acid residues that are not essential for activity. Suchproteins differ in amino acid sequence from SEQ ID NO:1 yet retainbiological activity. An isolated nucleic acid molecule encoding aprotein having a sequence that differs from that of SEQ ID NO:2 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence of SEQ ID NO:1.

Mutations can be introduced by standard techniques such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue with asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. For example, families include aminoacids with basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, trytophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenyalanine, tryptophan,histidine). The sequence analysis of the guinea pig receptor providedbelow in “Example 3” that compares the sequence of the guinea pig tohuman, rat and mouse provides guidance in the selection of non-essentialamino acids. Thus, a predicted nonessential amino acid residue wouldpreferably be replaced with another amino acid residue from the sameside chain family. Alternatively, mutations can be introduced randomlyalong the coding region or portions thereof, such as by saturationmutagenesis, and the resulting mutants screened for biological activityto identify mutants that retain activity. Following mutagenesis, theencoded protein can be expressed recombinantly and the activity of theprotein determined. In a preferred embodiment, the mutant protein can beassayed for the ability to form protein:protein interactions such aswith proteins in the prostanoid signaling pathway; the ability to bindligands such as ligands that bind to the prostanoid receptor; or, theability to bind to intracellular target proteins. The present inventionalso relates to native or mutant proteins or protein fragments ofdiagnostic, therapeutic or prophylactic use and would be useful forscreening for agonists, antagonists or modulators of receptor function.

Nucleotide sequences coding for a peptide may be altered so as to codefor a protein having properties that are different than those of thenaturally occurring peptide, such as changing the affinity of the ligandbinding domain or modulating the signal transduction pathway. Thepresent invention also relates to alterations of the nucleic acidsequence of SEQ ID NO:1 or portions thereof that modify the biologicalactivity of the protein.

Hybridization of Isolated Nucleic Acid Molecules

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to another nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. Low stringency hybridization conditions correspond to aT_(m) of 55° C. (e.g., 5× sodium chloride/sodium citrate (SSC), 0.1%SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m), (e.g., 40% formamide, with 5× or 6×SSC). High stringencyhybridization conditions correspond to the highest T_(m), (e.g., 50%formamide, 5× or 6×SSC). Hybridization requires that the two nucleicacids contain complementary sequences, although depending on thestringency of the hybridization, mismatches between bases are possible.The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-9.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8). A minimum length for a hybridizable nucleic acid molecule isat least about 20 nucleotides; particularly at least about 30nucleotides; more particularly at least about 40 nucleotides, even moreparticularly about 50 nucleotides, and yet more particularly at leastabout 60 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C.

In a particular embodiment of the present invention, a hybridizablenucleic acid molecule of the invention is at least 300, 325, 350, 375,400, 425, 450, 500, 550, 600, 650, 700, 800, 900 or 1000 nucleotides inlength and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence, preferably the codingsequence, of SEQ ID NO:1 a complement thereof, or a fragment thereof.The term “hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 55%, 60%, 65%, 70% and preferably 75% or morecomplementary to each other typically remain hybridized. Such stringentconditions are known to those skilled in the art and can be found in“Current Protocols in Molecular Biology”, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1 orthe complement thereof corresponds to a naturally occurring nucleic acidmolecule. As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein). One skilled in theart will appreciate that the conditions may be modified in view ofsequence-specific variables (e.g., length, G-C richness etc.). Inanother embodiment, an isolated nucleic acid molecule of the inventionthat hybridizes under stringent conditions to a potion of the sequenceof SEQ ID NO:1 can be used as a probe or a primer. The probe/primertypically comprises substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250,300, 350 or 400 consecutive nucleotides of the sense or anti-sensesequence of SEQ ID NO:1 or of a naturally occurring mutant of SEQ IDNO:1.

Antisense Nucleic Acid Molecules

The present invention encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein (e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence). An antisensenucleic acid can hydrogen bond to a sense nucleic acid. The antisensenucleic acid can be complementary to an entire nucleic acid sequence ofSEQ ID NO:1 or a portion thereof. Given the coding strand sequencesdisclosed herein (e.g., SEQ ID NO:1), antisense nucleic acids of theinvention can be designed according to the rules of Watson & Crick basepairing. An antisense oligonucleotide can be, for example, about 5, 10,15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be synthesized chemically using naturally occurringnucleotides or various chemically modified nucleotides designed toincrease the biological stability of the molecules, or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives, phosphonatederivatives and acridine-substituted nucleotides can be used.

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine,inosine, N⁶-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N⁶-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,β-D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid,wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil and2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically by using an expression vector into which thenucleic acid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest).

The antisense nucleic acid molecules of the invention typically areadministered to a subject or generated in situ so as to hybridize withor bind to cellular mRNA and/or genomic DNA encoding the protein of theinvention, thereby inhibiting expression of the protein by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecules that bind toDNA duplexes, through specific interactions in the major groove of thedouble helix, or to a regulatory region.

An example of a route of administration of antisense nucleic acidmolecules of the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense molecules can be modified suchthat the molecules specifically bind to receptors or antigens expressedon a selected cell surface, e.g., by linking the antisense nucleic acidmolecules to peptides or antibodies that bind to cell surface receptorsor antigens. The antisense nucleic acid molecules also can be deliveredto cells using the vectors described herein. To achieve sufficientintracelluar concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in that thestrands run parallel to each other (Gaultier et al., Nucleic Acids Res(1987)15:6625-6641). The antisense nucleic acid molecule also cancomprise a methylribonucleotide (Inoue et al., Nucleic Acids Res (1987)15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Left(1987) 215:327-330).

Ribozymes

The invention also encompasses ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, that hybridizes to theribozyme. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaselhoff et al., Nature (1988) 334:585-591)) can be used tocatalytically cleave nucleic acid transcripts and thus inhibittranslation of mRNA corresponding to SEQ ID NO:1. A ribozyme havingspecificity for the nucleic acid of SEQ ID NO:1 can be designed based onthe nucleotide sequence of SEQ ID NO:1. For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed so that the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved based on the reported nucleic acid sequence of SEQ ID NO:1(U.S. Pat. Nos. 4,987,071 and 5,116,742, the disclosures of which areincorporated by reference). Alternatively, the nucleic acid sequence ofSEQ ID NO:1 can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. (Bartel et al.,Science (1993) 261:1411-1418.

Triple Helical Nucleic Acid Molecules and Peptide Nucleic Acids

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofSEQ ID NO:1 (e.g., the promoter and/or enhancer region) to form triplehelical structures that prevent transcription of the gene in targetcells, see generally, Helene, Anticancer Drug Des (1991) 6(6):569;Helene Ann NY Acad Sci (1992) 660:27; and Maher, Bioassays (1992)14(12):807.

In particular embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety or phosphate backboneto improve, e.g., the stability, hybridization or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal., Bioorganic & Medicinal Chemistry (1996) 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in that the deoxyribose phosphate backbone is replacedby a pseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup et al.(1996) supra; Perry-O'Keefe et al., Proc Natl Acad Sci USA (1996)93:14670.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs ofthe present invention also can be used. For example, a PNA can be usedin the analysis of single base pair mutations in a gene by, e.g.,PNA-directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (Hyrup et al.(1996) supra) or as probes or primers for DNA sequence and hybridization(Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of the present invention can be modified,e.g., to enhance stability, specificity or cellular uptake, by attachinglipophilic or other helper groups to the PNA, by the formation ofPNA-DNA chimeras or by the use of liposomes or other techniques of drugdelivery known in the art. The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup et al. (1996) supra, Finn et al.,Nucleic Acids Res (1996) 24(17):3357-63, Mag et al., Nucleic Acids Res(1989) 17:5973; and Peterser et al., Bioorganic Med Chem Lett (1975)5:1119.

RNA/Nucleic Acid Interference

RNA interference (RNAi) or nucleic acid interference (NAi) is a processof sequence-specific post-transcriptional gene silencing mediated byshort interfering RNAs (siRNAs) or short interfering nucleic acids(siNA). This process is thought to be an evolutionarily conserveddefense mechanism whereby the production of double-stranded RNAs(dsRNAs) or double stranded nucleic acids (dsNA), for instance as aresult of viral infection, stimulates the activity of a ribonuclease IIIenzyme referred to as dicer (Berstein et al., 2001, Nature 409:363). Forinstance, Dicer processes the dsRNA into siRNA. Dicer may be involved inexcising 21- and 22-nucleotide small temporal RNAs (siRNAs) implicatedin translational control. The RNAi response also involves anendonuclease complex, an RNA-induced silencing complex (RISC), thatcleaves target single-stranded RNA having sequence complementary to theantisense strand of the siRNA (Elbashir et al., 2001, Genes Dev.,15:188). Optimal design of siRNAs, dsRNAs, siNAs or dsNAs based onlength, structure, chemical composition and sequence for efficient RNAior NAi are known to those skilled in the art (for examples see: Chiu andRana et al., 2003, RNA 9:1034-48; Elbashir et al., 2001; Parish et al.,2000; PCT Publication Nos., WO 03/070744, WO 01/75164, WO 01/68836, WO01/49844, WO 01/36646, WO 01/29058, WO 00/44914 WO 00/01846, WO99/32619,WO 99/07409 WO 99/53050; Canadian Patent Application No.2,359,180, the disclosures of which are incorporated by reference). Somepossible modifications to the siNA or dsNA to improve activity includebut are not limited to: 3′-terminal dinucleotide overhangs, substitutionof one or both siNA strands with 2′-deoxy nucleotides (2′-H), replacingthe 3′-terminal nucleotide overhanging segments of the siNA duplex withdeoxyribonucleotides, modifications to either the phosphate-sugarbackbone or the nucleoside to include at least one of a nitrogen orsulfur heteroatom, 2′-amino or 2′-O-methyl nucleotides and nucleotidescontaining a 2′-O or 4′-C methylene bridge in dsRNA constructs,substituting 4-thiouracil, 5-bromouracil, 5-iodouracil and3-(aminoallyl)uracil in sense and antisense strands. PCT Publication No.WO 01/68836 describes methods for using endogenously derived dsRNA toattenuate gene expression. Further, the mRNA targeted for RNAi has beensuggested to act as a template for 5′ to 3′ synthesis of new dsRNAtargeted to a gene in one cell type and can lead to RNAi-mediatedsilencing of a second gene expressed in a distinct cell type, aphenomenon termed transitive RNAi (Alder et al., 2003 rna 9:25).

Protein

The present invention extends to an isolated polypeptide comprising theamino acid sequence of SEQ ID NO:2, a variant thereof, a fragmentthereof or an analog or derivative thereof.

An isolated nucleic acid molecule encoding a protein of the presentinvention having a sequence that differs from that of SEQ ID NO:2, e.g.a variant, can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO:I such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. For example, thefirst and third intracellular loops are three and five amino acidsshorter in the guinea pig DP protein, respectively, whereas in themouse, human and rat DP proteins these intracellular loops are all ofidentical size. A variant of SEQ ID NO:2 could be created be insertingone or more nucleotides found in any of the other orthologues.

In a particular embodiment, a mutant protein of the present inventioncan be assayed for: (1) the ability to form protein:protein interactionswith proteins in the signaling pathway; (2) the ability to bind aligand; (3) the ability to bind to an intracellular target protein, or(4) the ability to modulate cellular proliferation, cellulardifferentiation or cellular response.

Native proteins of the invention can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. Alternatively, proteins of the invention canreadily be produced by recombinant DNA techniques. Yet anotheralternative embodiment, is the chemical synthesis of the protein or thepolypeptide of the invention using standard peptide synthesistechniques.

Biologically active portions or fragments of a protein of the inventioninclude peptides comprising amino acid sequences sufficiently identicalto or derived from the amino acid sequence of SEQ ID NO:2, that includefewer amino acids than the full length protein of the invention andexhibit at least one activity of the protein of the invention.Typically, biologically active portions comprise a domain or motif withat least one activity of the protein of the invention. For instance, abiologically active fragment of the protein of the invention couldcontain two sequence stretches that have previously been reported to becharacteristically conserved amongst GPCRs of the prostanoid family(Hirata et al., 1994) and are also present in the guinea pig DP protein:

QYCPGTWCR (SEQ ID NO: 10) in the second extracellular loop andRFLSVISIVDPWIFI (SEQ ID NO: 11) in the seventh transmembrane domain. Abiologically active portion of the protein of the invention can be apolypeptide that is, for example, 10, 25, 50, 100 or more amino acids inlength. Particular biologically active polypeptides include one or moreidentified structural domains of the protein of the present invention.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of the protein ofthe invention. Further guidance directed to biologically relevantportion of the invention are provided below in “Example 3”.

Other useful proteins are substantially identical to SEQ ID NO:2 andretain a functional activity of the protein of SEQ ID NO:2 yet differ inamino acid sequence due to natural allelic variation or mutagenesis. Forexample, such proteins and polypeptides possess at least one biologicalactivity described herein. Accordingly, a useful protein of theinvention is a protein that includes an amino acid sequence at leastabout 65%, 75%, 85%, 95%, 99% or 100% identical to the amino acidsequence of SEQ ID NO:2 and retains a functional activity of the proteinof SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions then arecompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are considered identical at thatposition. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A particular, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin et al., Proc Natl Acad Sci USA(1990) 87:2264, modified as in Karlin et al., Proc Natl Acad Sci USA(1993) 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al., J Mol Bio (1990) 215:403. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., Nucleic Acids Res (1997)25:3389. Alternatively, PSI-Blast can be used to perform an iteratedsearch that detects distant relationships between molecules. Altschul etal. (1997) supra. When utilizing BLAST, Gapped BLAST and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used, see http://www.ncbi.nim.nih.gov. Anotherparticular, non-limiting example of a mathematical algorithm utilizedfor the comparison of sequences is the algorithm of Myers et al., CABIOS(1988) 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0) that is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12 anda gap penalty of 4 may be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The present invention further extends to chimeric or fusion proteins ofthe invention. As used herein, a “chimeric protein” or “fusion protein”of the invention comprises a polypeptide of SEQ ID NO:2 operably linkedto a “polypeptide not of the invention”. A “polypeptide of theinvention” refers to a polypeptide having an amino acid sequencecorresponding to SEQ ID NO:2. A “polypeptide not of the invention”refers to a polypeptide having an amino acid sequence corresponding to aprotein that is not substantially identical to SEQ ID NO:2, e.g., aprotein that is different from the protein of the invention and isderived from the same or a different organism. Within a fusion proteinof the invention, the polypeptide of the invention can correspond to allor a portion of a SEQ ID NO:2, preferably at least one biologicallyactive portion of a SEQ ID NO:2. Within the fusion protein, the term“operably linked” is intended to indicate that the polypeptide of theinvention and the polypeptide not of the invention are fused in-frame toeach other. The polypeptide not of the invention can be fused to theN-terminus or C-terminus of the polypeptide of the invention. One usefulfusion protein utilizes glutathione-S-transferase (GST) in which thepolypeptide of the invention is fused to the C-terminus of GST. Suchfusion proteins can facilitate the purification of recombinantpolypeptides of the invention.

In another embodiment, a fusion protein of the present invention extendsto an immunoglobulin fusion protein in that all or part of SEQ ID NO:2is fused to sequences derived from a member of the immunoglobulinprotein family. The immunoglobulin-fusion protein of the invention canbe incorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a ligand and the receptorprotein of the invention on the surface of a cell, thereby to suppressreceptor-mediated signal transduction in vivo. The immunoglobulin-fusionprotein of the invention can be used to affect the bioavailability of acognate ligand of the receptor of the present invention. Inhibition ofthe ligand-receptor interaction may be useful therapeutically, such asbut not limited to, treating or modulating sleep, body temperature,olfactory function, hormone release, pain, gastrointestinal tractdisorders, liver disease, eye diseases such as glaucoma, blood disorderssuch as thrombosis, inflammatory disorders including but not limited toasthma, allergic rhinitis, airway hyperactivity, allergic dermatitis,allergic conjunctivitis and chronic obstructive pulmonary disease.Moreover, the immunoglobulin-polypeptide fusion proteins of theinvention can be used as immunogens to produce antibodies in a subject,to purify ligands and in screening assays to identify molecules thatinhibit the interaction of the receptor of the invention with a ligand.

In a particular embodiment, a chimeric or fusion protein of the presentinvention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example, by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that subsequently canbe annealed and reamplified to generate a chimeric gene sequence (seee.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding the polypeptide of the inventionor a portion thereof can be cloned into such an expression vector sothat the fusion moiety is linked in-frame to the protein of theinvention.

Nucleic Acid and Protein Variants

As explained above, the present invention further extends to variants ofSEQ ID NO:1 and SEQ ID NO:2. For example, mutations may be introducedinto the amino acid sequence of SEQ ID NO:1 using standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Moreover, conservative amino acid substitutions can be made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. For example, oneor more amino acids can be substituted by another amino acid of asimilar polarity, which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the amino acidsequence of a polypeptide of the present invention may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.Amino acids containing aromatic ring structures are phenylalanine,tryptophan, and tyrosine. The polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Such alterations will not be expected to effectapparent molecular weight as determined by polyacrylamide gelelectrophoresis, or isoelectric point.

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.        Additional substitutions may be made with synthetic (i.e.,        non-naturally occurring) amino acids.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced for a potential site for disulfide bridges with another Cys.A H is may be introduced as a particularly “catalytic” site (i.e., Hiscan act as an acid or base and is the most common amino acid inbiochemical catalysis). Pro may be introduced because of itsparticularly planar structure, which induces β-turns in the protein'sstructure.

Mutations can also be introduced randomly along all or part of a codingsequence of SEQ ID NO:1, such as by saturation mutagenesis, and theresultant mutants can be screened for biological activity to identifymutants that retain activity. Following mutagenesis, the encoded proteincan be expressed recombinantly and the activity of the protein can bedetermined.

Variants of the present invention can function as an agonist (mimetic)or as an antagonist. Variants of the protein of the invention can begenerated by mutagenesis, e.g., discrete point mutation or truncation ofthe protein of the invention. An agonist of the protein of the inventioncan retain substantially the same or a subset of the biologicalactivities of the naturally occurring protein of the invention. Anantagonist can competitively bind to a downstream or upstream member ofa cellular signaling cascade that includes the protein of the invention,and thus inhibit one or more of the activities of the naturallyoccurring form of the protein of the invention. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. Treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein of the invention can have fewer side effects in a subjectrelative to treatment with the naturally occurring form of the protein.

Variants of the protein of the invention that function as eitheragonists (mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants of the protein of theinvention are generated by combinatorial mutagenesis at the nucleic acidlevel, and is encoded by a variegated gene library. A variegated libraryof variants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential nucleic acid sequences of the invention areexpressed as individual polypeptides or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofsequences of the invention therein. There are a variety of methods thatcan be used to produce libraries of potential variants of the inventionfrom a degenerate oligonucleotide sequence. Chemical synthesis of adegenerate gene sequence can be performed in an automated DNAsynthesizer and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential nucleic acid sequences of the invention. Methods forsynthesizing degenerate oligonucleotides are known in the art (see,e.g., Narang, Tetrahedron (1983) 39:3; Itakura et al., Ann Rev Biochem(1984) 53:323; Itakura et al., Science (1984) 198:1056; Ike et al.,Nucleic Acid Res (1983) 11:477).

In addition, libraries of fragments of the protein coding sequence canbe used to generate a variegated population of fragments for screeningand subsequent selection of variants of a protein of the invention. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double-stranded PCR fragment of a coding sequence of theinvention with a nuclease under conditions wherein nicking occurs onlyabout once per molecule, denaturing the double-stranded DNA, renaturingthe DNA to form double-stranded DNA that can include sense/antisensepairs from different nicked products, removing single-stranded portionsfrom reformed duplexes by treatment with S1 nuclease and ligating theresulting fragment library into an expression vector. By that method, anexpression library can be derived that encodes N-terminal and internalfragments of various sizes of the protein of the invention.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of the protein of theinvention. The most widely used techniques that are amenable to highthrough-put analysis for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique that enhances the frequency of functionalmutants in the libraries, can be used in combination with the screeningassays to identify variant proteins of the invention (Arkin et al., ProcNatl Acad Sci USA (1992) 89:7811-7815; Delgrave et al., ProteinEngineering (1993) 6(3):327-331).

Analogs and Derivatives of the Protein of the Invention

Moreover, the present invention also includes derivatives or analogs ofthe protein of the invention produced from a chemical modification. Aprotein of the present invention may be derivatized by the attachment ofone or more chemical moieties to the protein moiety.

The chemical moieties suitable for derivatization may be selected fromamong water soluble polymers so that the analog or derivative does notprecipitate in an aqueous environment, such as a physiologicalenvironment. Optionally, the polymer will be pharmaceuticallyacceptable. One skilled in the art will be able to select the desiredpolymer based on such considerations as whether the polymer/componentconjugate will be used therapeutically, and if so, the desired dosage,circulation time, resistance to proteolysis, and other considerations.For the protein of the invention, these may be ascertained using theassays provided herein. Examples of water soluble polymers havingapplications herein include, but are not limited to, polyethyleneglycol, copolymers of ethylene glycol/propylene glycol,carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), dextran, poly(-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols or polyvinyl alcohol. Polyethyleneglycol propionaldenhyde may have advantages in manufacturing due to itsstability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects if any, on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached to the protein of theinvention may vary, and one skilled in the art will be able to ascertainthe effect on function. One may mono-derivatize, or may provide for adi-, tri-, tetra- or some combination of derivatization, with the sameor different chemical moieties (e.g., polymers, such as differentweights of polyethylene glycols). The proportion of polymer molecules toprotein molecules of the invention will vary, as will theirconcentrations in the reaction mixture. In general, the optimum ratio(in terms of efficiency of reaction in that there is no excess unreactedcomponent or components and polymer) will be determined by factors suchas the desired degree of derivatization (e.g., mono, di-, tri-, etc.),the molecular weight of the polymer selected, whether the polymer isbranched or unbranched, and the reaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein of the invention with consideration of effectson functional or antigenic domains. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0401384 hereinincorporated by reference (coupling PEG to G-CSF), see also Malik etal., 1992, Exp. Hematol. 20:1028-1035 (reporting pegylation of GM-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group include lysine residues and theN-terminal amino acid residues; those having a free carboxyl groupinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecule(s).Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

One may specifically desire a N-terminally chemically modified proteinof the invention. Using polyethylene glycol as an illustration of thepresent compositions, one may select from a variety of polyethyleneglycol molecules (by molecular weight, branching, etc.), the proportionof polyethylene glycol molecules to protein molecules of the inventionin the reaction mix, the type of pegylation reaction to be performed,and the method of obtaining the selected N-terminally pegylated protein.The method of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization. Under theappropriate reaction conditions, substantially selective derivatizationat the N-terminus with a carbonyl group containing polymer is achieved.For example, one may selectively N-terminally pegylate the protein ofthe invention by performing the reaction at a pH which allows one totake advantage of the pK_(a) differences between the ε-amino groups ofthe lysine residues and that of the α-amino group of the N-terminalresidue. By such selective derivatization, attachment of a water solublepolymer to the protein of the invention is controlled: the conjugationwith the polymer takes place predominantly at the N-terminus and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs. Using reductive alkylation, the watersoluble polymer may be of the type described above, and should have asingle reactive aldehyde for coupling to the protein of the invention.Polyethylene glycol proprionaldehyde, containing a single reactivealdehyde, may be used.

Antibodies

An isolated protein of the invention or a portion or fragment thereof,can be used as an immunogen to generate antibodies that bind the proteinof the invention using standard techniques for polyclonal and monoclonalantibody preparation. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site that is specific for—that is, that binds to—anantigen, such as the protein of the invention, or a fragment thereof. Amolecule that specifically binds to the protein of the invention is amolecule that binds the protein of the invention, but does notsubstantially bind other molecules in a sample, e.g., a biologicalsample that naturally contains the protein of the invention. Examples ofimmunologically active portions of immunoglobulin molecules includeF_((ab)) and F_((ab′)2) fragments that can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal, monoclonal and chimeric antibodies that have the protein ofthe invention, a variant thereof, a fragment thereof, or an analog orderivative thereof, as an immunogen. Chimeric antibodies are preferredfor use in therapy of human diseases or disorders, since the human orhumanized antibodies are much less likely than xenogenic antibodies toinduce an immune response, in particular an allergic response,themselves.

The full-length protein of the invention can be used or, alternatively,the invention provides antigenic peptide fragments of the invention foruse as immunogens. The antigenic peptide of the invention comprises atleast 8 (preferably 10, 15, 20, 30 or more) amino acid residues of theamino acid sequence shown in SEQ ID NO:2 and encompasses an epitope suchthat an antibody raised against the peptide forms a specific immunecomplex with the protein of the invention.

An immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed protein of the invention or achemically synthesized polypeptide of the invention. The preparationfurther can include an adjuvant, such as Freund's complete or incompleteadjuvant or similar immunostimulatory agent. Immunization of a suitablesubject with an immunogenic preparation induces a polyclonal antibodyresponse directed against the protein of the invention.

An antibody of the present invention can be a monoclonal antibody, apolyclonal antibody, or a chimeric antibody. The term “monoclonalantibody” or “monoclonal antibody composition”, as used herein, refersto a population of antibody molecules that contain only one species ofan antigen-binding site capable of immunoreacting with a particularepitope of the protein of the invention. A monoclonal antibodycomposition thus typically displays a single binding affinity for aparticular epitope of the protein of the invention.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with an immunogen of the invention. The antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme-linked immunosorbent assay (ELISA)using the protein of the invention that has been immobilized. Ifdesired, the antibody molecules directed against the protein of theinvention can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography, to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler et al., Nature (1975)256:495-497, the human B cell hybridoma technique (Kohler et al.,Immunol Today (1983) 4:72), the EBV hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, (1985), Alan R. Liss, Inc.,pp. 77-96) or trioma techniques. The technology for producing hybridomasis well known (see generally Current Protocols in Immunology (1994)Coligan et al., eds., John Wiley & Sons, Inc., New York, N.Y.). Briefly,an immortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with an immunogen of theinvention as described above and the culture supernatants of theresulting hybridoma cells are screened to identify a hybridoma producinga monoclonal antibody that binds the protein of the invention.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody (see, e.g., Current Protocols in Immunology, supra;Galfre et al., Nature (1977) 266:550-552; Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); and Lerner, Yale J Biol Med (1981)54:387-402). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods that also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line, e.g., a myeloma cell line that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/I-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. The myelomalines are available from ATCC. Typically, HAT-sensitive mouse myelomacells are fused to mouse splenocytes using polyethylene glycol (“PEG”).Hybridoma cells resulting from the fusion then are selected using HATmedium that kills unfused and unproductively fused myeloma cells(unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind the protein of the invention, e.g., using astandard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the protein of the invention thereby toisolate immunoglobulin library members that bind the protein of theinvention. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene “SURFZAP” PhageDisplay Kit, Catalog No. 240612).

Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display libraries are known tothose skilled in the art. (for example, Fuchs et al., Bio/Technology(1991)9:1370 1372; Hay et al., Hum Antibody Hybridomas (1992) 3:81 85;Huse et al., Science (1989) 246:1275-1281; Griffiths et al., EMBO J.(1993) 25(12):725-734; U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809, the disclosures of which areincorporated by reference).

Furthermore, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies comprising both human and non-human portions, canbe made using standard recombinant DNA techniques. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art (for example using methods described in PCTPublication No. WO 87/02671; Europe Patent Application No. 184,187;Europe Patent Application No. 171,496; Europe Patent Application No.173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;Europe Patent Application No. 125,023; Better et al., Science (1988)240:1041-1043; Liu et al., Proc Natl Acad Sci USA (1987) 84:3439-3443;Lin et al., J Immunol (1987) 139:3521-3526; Sun et al., Proc Natl AcadSci USA (1987) 84:214-218; Nishimura et al., Canc Res (1987)47:999-1005; Wood et al., Nature (1985) 314:446-449; Shaw et al., J NatlCancer Inst (1988) 80:1553-1559; Morrison, Science (1985) 229:1202-1207;Oi et al., Bio/Techniques (1986) 4:214; U.S. Pat. No. 5,225,539; Joneset al., Nature (1986) 321:552-525; Verhoeyan et al., Science (1988)239:1534; and Beidler et al., J Immunol (1988) 141:4053-4060; thedisclosures of which are incorporated by reference).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but can express human heavyand light chain genes. The transgenic mice are immunized in the normalfashion with a selected antigen, e.g., all or a portion of the proteinof the invention. Monoclonal antibodies directed against the antigen canbe obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation and subsequently undergo class switchingand somatic mutation. Thus, using such an epitope, e.g., an antibodythat inhibits the activity of the protein of the invention isidentified. The heavy chain and the light chain of the non-humanantibody are cloned and used to create phage display F_(ab) fragments.For example, the heavy chain gene can be cloned into a plasmid vector sothat the heavy chain can be secreted from bacteria. The light chain genecan be cloned into a phage coat protein gene so that the light chain canbe expressed on the surface of phage. A repertoire (random collection)of human light chains fused to phage is used to infect the bacteria thatexpress the non-human heavy chain. The resulting progeny phage displayhybrid antibodies (human light chain/non-human heavy chain). Theselected antigen is used in a panning screen to select phage that bindthe selected antigen. Several rounds of selection may be required toidentify such phage.

Human light chain genes are isolated from the selected phage that bindthe selected antigen. The selected human light chain genes then are usedto guide the selection of human heavy chain genes as follows. Theselected human light chain genes are inserted into vectors forexpression by bacteria. Bacteria expressing the selected human lightchains are infected with a repertoire of human heavy chains fused tophage. The resulting progeny phage display human antibodies (human lightchain/human heavy chain).

Next, the selected antigen is used in a panning screen to select phagethat bind the selected antigen. The selected phage display a completelyhuman antibody that recognizes the same epitope recognized by theoriginal selected, non-human monoclonal antibody. The genes encodingboth the heavy and light chains are isolated and can be manipulatedfurther for production of human antibody. The technology is described byJespers et al. (Bio/Technology (1994) 12:899-903).

An antibody (e.g., monoclonal antibody) can be used to isolate theprotein of the invention by standard techniques, such as affinitychromatography or immunoprecipitation. An antibody directed against theprotein of the invention can facilitate the purification of the naturalprotein from cells and of recombinantly produced protein expressed inhost cells. Moreover, an antibody can be used to detect the protein ofthe invention (e.g., in a cellular lysate or cell supernatant) toevaluate the abundance and pattern of expression of the protein.Antibodies can be used diagnostically to monitor protein levels intissue as part of a clinical testing procedure, for example, todetermine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance, whichare described infra.

Detectable Labels

Optionally, isolated nucleic acid molecules of the present invention,polypeptides of the present invention, and antibodies of the presentinvention, as well as fragments of such moieties, may be detectablylabeled. Suitable labels include enzymes, fluorophores (e.g.,fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR),rhodamine, free or chelated lanthanide series salts, especially Eu³⁺, toname a few fluorophores), chromophores, radioisotopes, chelating agents,dyes, colloidal gold, latex particles, ligands (e.g., biotin),bioluminescent materials, and chemiluminescent agents. When a controlmarker is employed, the same or different labels may be used for thereceptor and control marker.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

Direct labels are one example of detectable labels that can be usedaccording to the present invention. A direct label has been defined asan entity, which in its natural state, is readily visible, either to thenaked eye, or with the aid of an optical filter and/or appliedstimulation, e.g. U.V. light to promote fluorescence. Among examples ofcolored labels, which can be used according to the present invention,include metallic sol particles, for example, gold sol particles such asthose described by Leuvering (U.S. Pat. No. 4,313,734); dye soleparticles such as described by Gribnau et al. (U.S. Pat. No. 4,373,932)and May et al. (WO 88/08534); dyed latex such as described by May,supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated inliposomes as described by Campbell et al. (U.S. Pat. No. 4,703,017).Other direct labels include a radionucleotide, a fluorescent moiety or aluminescent moiety. In addition to these direct labelling devices,indirect labels comprising enzymes can also be used according to thepresent invention. Various types of enzyme linked immunoassays are wellknown in the art, for example, alkaline phosphatase and horseradishperoxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactatedehydrogenase, urease, these and others have been discussed in detail byEva Engvall in Enzyme immunoassay ELISA and EMIT in Methods inEnzymology, 70. 419-439, 1980 and in U.S. Pat. No. 4,857,453.

Other detectable labels for use in the invention include magnetic beadsor magnetic resonance imaging labels.

In another embodiment, a phosphorylation site can be created on anisolated polypeptide of the present invention, an antibody of thepresent invention, or a fragment thereof, for labeling with ³²P, e.g.,as described in European Patent No. 0372707.

As exemplified herein, proteins, including antibodies, can be detectablylabeled by metabolic labeling. Metabolic labeling occurs during in vitroincubation of the cells that express the protein in the presence ofculture medium supplemented with a metabolic label, such as[³⁵S]-methionine or [³²P]-orthphosphate. In addition to metabolic (orbiosynthetic) labeling with [³⁵S]-methionine, the invention furthercontemplates labeling with [¹⁴C]-amino acids and [³H]-amino acids (withthe tritium substituted at non-labile positions).

Antibodies may further be detected using, in addition to the labelrecited above, antigenic peptide tags recognizable by antibodies.Examples include HA tags and FLAG® tags.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding SEQ ID NO:1 or aportion thereof. As explained above, one type of vector is a “plasmid,”which refers to a circular double-stranded DNA loop into whichadditional DNA segments can be ligated. Another type of vector is aviral vector, wherein additional DNA segments can be ligated into aviral genome. Certain vectors are capable of autonomous replication in ahost cell (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell on introduction into the host cell and thereby are replicated alongwith the host genome. Moreover, expression vectors are capable ofdirecting the expression of genes operably linked thereto. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids (vectors). However, the invention is intended toinclude such other forms of expression vectors, such as viral vectors(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), that serve equivalent functions.

A recombinant expression vector of the invention comprises a nucleicacid molecule of the present invention in a form suitable for expressionof the nucleic acid in a host cell. That means a recombinant expressionvector of the present invention includes one or more regulatorysequences, selected on the basis of the host cells to be used forexpression, that is operably linked to the nucleic acid to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology Vol. 185,Academic Press, San Diego, Calif. (1990). Regulatory sequences includethose that direct constitutive expression of the nucleotide sequence inmany types of host cells (e.g., tissue specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of hostcell to be transformed, the level of expression of protein desired etc.The expression vectors of the invention can be introduced into hostcells to produce proteins or peptides encoded by nucleic acids asdescribed herein

A recombinant expression vector of the invention can be designed forexpression of SEQ ID NO:1 or a portion thereof in prokaryotic oreukaryotic cells, e.g., bacterial cells such as E. coli, insect cells(using baculovirus expression vectors), yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using phage regulatory elements andproteins, such as, a T7 promoter and/or a T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes and the cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith et al., Gene (1988) 67:31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRITS (Pharmacia, Piscataway, N.J.), that fuse glutathione5-transferase (GST), maltose E binding protein or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology, AcademicPress, San Diego, Calif. (1990) 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host with impaired capacity to cleaveproteolytically the recombinant protein (Gottesman, Gene ExpressionTechnology: Methods in Enzymology, Academic Press, San Diego, Calif.(1990) 185:119-128). Another strategy is to alter the nucleic acidsequence of the nucleic acid molecule to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., Nucleic Acids Res(1992) 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector of the invention is a yeastexpression vector. Examples of vectors for expression in yeast such asS. cerevisiae include pYepSecI (Baldari et al., EMBO J. (1987)6:229-234), pMFa (Kurjan et al., Cell (1982) 30:933-943), pJRY88(Schultz et al., Gene (1987) 54:113-123), pYES2 (Invitrogen Corporation,San Diego, Calif.) and pPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, SEQ ID NO:1 or a portion thereof can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol Cell Biol (1983)3:2156-2165) and the pVL series (Lucklow et al., Virology (1989)170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors having applications herein include, butcertainly are not limited to pCDM8 (Seed, Nature (1987) 329:840) andpMT2PC (Kaufman et al., EMBO J. (1987) 6:187-195). When used inmammalian cells, control functions of the expression vector often areprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, a recombinant mammalian expression vector of thepresent invention is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al., Genes Dev (1987) 1:268-277),lymphoid-specific promoters (Calame et al., Adv Immunol (1988)43:235-275), in particular, promoters of T cell receptors (Winoto etal., EMBO J (1989) 8:729-733) and immunoglobulins (Banerji et al., Cell(1983) 33:729-740; Queen et al., Cell (1983) 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne etal., Proc Natl Acad Sci USA (1989) 86:5473-5477), pancreas-specificpromoters (Edlund et al., Science (1985) 230:912-916) and mammarygland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.4,873,316 and Europe Application No. 264,166). Developmentally-regulatedpromoters also are encompassed, for example the murine hox promoters(Kessel et al., Science (1990) 249:374-379) and the α-fetoproteinpromoter (Campes et al., Genes Dev (1989) 3:537-546). The disclosures ofeach of the foregoing references are incorporated herein by reference.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into an expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to mRNA of the invention. Regulatory sequencesoperably linked to a nucleic acid cloned in the antisense orientationcan be chosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types. For example, viral promoters and/orenhancers or regulatory sequences can be chosen that directconstitutive, tissue-specific or cell type-specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes, see Weintraub etal. (Reviews-Trends in Genetics, Vol. 1(1)1986).

Another aspect of the present invention pertains to host cells intowhich a recombinant expression vector of the invention has beenintroduced. The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but also to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but still are included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, theprotein of the invention can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO), 293 cells or COS cells). Other suitable host cellsare known to those skilled in the art. Vector DNA can be introduced intoprokaryotic or eukaryotic cells via conventional transformation ortransfection techniques. As used herein, the terms “transformation” and“transfection” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,transduction, DEAE-dextran-mediated transfection, lipofection orelectroporation.

For stable transfection of mammalian cells, it is known that, dependingon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into the genome. Toidentify and to select the integrants, a gene that encodes a selectablemarker (e.g., for resistance to antibiotics) generally is introducedinto the host cells along with the gene of interest. Preferredselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding SEQ ID NO:1 or a portion thereof or the nucleic acid encoding aselectable marker can be introduced on a separate vector. For example,cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) the protein ofthe invention. Accordingly, the invention further provides methods forproducing SEQ ID NO:2 or a portion thereof by using the host cells ofthe invention. In one embodiment, the method comprises culturing thehost cell of invention (into which a recombinant expression vectorencoding SEQ ID NO:1 has been introduced) in a suitable medium such thatthe protein of the invention is produced. In another embodiment, themethod further comprises isolating the protein of the invention from themedium or the host cell.

In another embodiment, the invention comprises an inducible expressionsystem for the recombinant expression of other proteins subcloned inmodified expression vectors. For example, host cells comprising amutated G protein (e.g., yeast cells, Y2 adrenocortical cells and cyc⁻S49, see U.S. Pat. Nos. 6,168,927 B1, 5,739,029 and 5,482,835; Mitchellet al., Proc Natl Acad Sci USA (1992) 89(19):8933-37 and Katada et al.,J Biol Chem (1984) 259(6):3586-95) are transduced with a firstexpression vector comprising a nucleic acid sequence encoding SEQ IDNO:1, wherein SEQ ID NO:2 is functionally expressed in the host cells.Even though the expressed protein of the invention is constitutivelyactive, the mutation does not allow for signal transduction; i.e., noactivation of a G-protein directed downstream cascade occurs (e.g., noadenylyl cyclase activation). Subsequently, a second expression vectoris used to transduce the SEQ ID NO:1-comprising host cells. The secondvector comprises a structural gene that complements the G proteinmutation of the host cell (i.e., functional mammalian or yeast G_(s),G_(i), G_(o), or G_(q), e.g., see PCT Publication No. WO 97/48820; U.S.Pat. Nos. 6,168,927 B1, 5,739,029 and 5,482,835 and which are herebyincorporated by reference herein in their entireties) in addition to thegene of interest to be expressed by the inducible system. Thecomplementary structural gene of the second vector is inducible; i.e.,under the control of an exogenously added component (e.g., tetracycline,IPTG, small molecules etc., see Sambrook et al. supra) that activates apromoter which is operably linked to the complementary structural gene.On addition of the inducer, the protein encoded by the complementarystructural gene is functionally expressed such that the constitutivelyactive protein of the invention now will form a complex that leads toappropriate downstream pathway activation (e.g., second messengerformation). The gene of interest comprising the second vector possessesan operably linked promoter that is activated by the appropriate secondmessenger (e.g., CREB, AP1 elements). Thus, as second messengeraccumulates, the promoter upstream from the gene of interest isactivated to express the product of said gene. When the inducer isabsent, expression of the gene of interest is switched off.

In a particular embodiment, the host cells for the inducible expressionsystem include, but are not limited to, S49 (cyc⁻) cells. While celllines are contemplated that comprise G-protein mutations, suitablemutants may be artificially produced/constructed (see U.S. Pat. Nos.6,168,927 B1, 5,739,029 and 5,482,835 for yeast cells).

In a related aspect, the cells are transfected with a vector operablylinked to a cDNA comprising a sequence encoding a protein as set forthin SEQ ID NO:2. The first and second vectors comprising said system arecontemplated to include, but are not limited to, pCDM8 (Seed, Nature(1987) 329:840) and pMT2PC (Kaufman et al., EMBO J (1987) 6:187-195),pYepSecI (Baldari et al., EMBO J (1987) 6:229-234), pMFa (Kurjan et al.,Cell (1982) 30:933-943), pJRY88 (Schultz et al., Gene (1987)54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.) and pPicZ(Invitrogen Corp, San Diego, Calif.).

In a related aspect, the host cells may be transfected by such suitablemeans, wherein transfection results in the expression of a functionalprotein (e.g., Sambrook et al., supra, and Kriegler, Gene Transfer andExpression: A Laboratory Manual, Stockton Press, New York, N.Y., 1990).Such “functional proteins” include, but are not limited to, proteinsthat once expressed, form complexes with G-proteins, where theG-proteins regulate second messenger formation. Other methods fortransfecting host cells that have applications herein include, butcertainly are not limited to transfection, electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, lipofection (lysosome fusion), use of a genegun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol.Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624;Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar.15, 1990).

A large variety of promoters have applications in the present invention.Indeed, expression of a polypeptide of the present invention may becontrolled by any promoter/enhancer element known in the art, but theseregulatory elements must be functional in the host selected forexpression. Promoters which may be used to control expression include,but are not limited to, the SV40 early promoter region (Benoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter; and the animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986,Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987,Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

Expression vectors containing a nucleic acid molecule of the inventioncan be identified by four general approaches: (a) PCR amplification ofthe desired plasmid DNA or specific mRNA, (b) nucleic acidhybridization, (c) presence or absence of selection marker genefunctions, and (d) expression of inserted sequences. In the firstapproach, the nucleic acids can be amplified by PCR to provide fordetection of the amplified product. In the second approach, the presenceof a foreign gene inserted in an expression vector can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to an inserted marker gene. In the third approach, therecombinant vector/host system can be identified and selected based uponthe presence or absence of certain “selection marker” gene functions(e.g., β-galactosidase activity, thymidine kinase activity, resistanceto antibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of foreign genes in thevector. In another example, if the nucleic acid encoding the protein ofthe invention, a variant thereof, or an analog or derivative thereof, isinserted within the “selection marker” gene sequence of the vector,recombinants containing the insert can be identified by the absence ofthe gene function. In the fourth approach, recombinant expressionvectors can be identified by assaying for the activity, biochemical, orimmunological characteristics of the gene product expressed by therecombinant vector, provided that the expressed protein assumes afunctionally active conformation.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors may consist, for example, of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol EI, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., 1988, Gene67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS,e.g., the numerous derivatives of phage λ, e.g., NM989, and other phageDNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmidssuch as the 2μ plasmid or derivatives thereof; vectors useful ineukaryotic cells, such as vectors useful in insect or mammalian cells;vectors derived from combinations of plasmids and phage DNAs, such asplasmids that have been modified to employ phage DNA or other expressioncontrol sequences; and the like.

For example, in a baculovirus expression systems, both non-fusiontransfer vectors, such as but not limited to pVL941 (BamH1 cloning site;Summers), pVL1393 (BamH1, SmaI, XbaI, EcoR1, NotI, XmaIII, Bg/II, andPstI cloning site; Invitrogen), pVL1392 (Bg/II, PstI, NotI, XmaIII,EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers and Invitrogen), andpBlueBacIII (BamH1, Bg/II, PstI, NcoI, and HindIII cloning site, withblue/white recombinant screening possible; Invitrogen), and fusiontransfer vectors, such as but not limited to pAc700 (BamH1 and KpnIcloning site, in which the BamH1 recognition site begins with theinitiation codon; Summers), pAc701 and pAc702 (same as pAc700, withdifferent reading frames), pAc360 (BamH1 cloning site 36 base pairsdownstream of a polyhedrin initiation codon; Invitrogen(195)), andpBlueBacHisA, B, C (three different reading frames, with BamH1, Bg/II,PstI, NcoI, and HindIII cloning site, an N-terminal peptide for ProBondpurification, and blue/white recombinant screening of plaques;Invitrogen (220) can be used.

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as the dihydrofolatereductase (DHFR) promoter, e.g., any expression vector with a DHFRexpression vector, or a DHFR/methotrexate co-amplification vector, suchas pED (PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12 (1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SbaI, EcoRI, and Bc/I cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV-LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindIII, NotI, XhoI, SfiI, BamH1 cloning site, inducible metallothioneinlia gene promoter, hygromycin selectable marker: Invitrogen), pREP8(BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloning site, RSV-LTRpromoter, histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI,HindIII, NotI, XhoI, SfiI, and BamHI cloning site, RSV-LTR promoter,G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter,hygromycin selectable marker, N-terminal peptide purifiable via ProBondresin and cleaved by enterokinase; Invitrogen). Selectable mammalianexpression vectors for use in the invention include pRc/CMV (HindIII,BstXI, NotI, SbaI, and ApaI cloning site, G418 selection; Invitrogen),pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning site, G418 selection;Invitrogen), and others. Vaccinia virus mammalian expression vectors(see, Kaufman, 1991, supra) for use according to the invention includebut are not limited to pSC11 (SmaI cloning site, TK- and β-galselection), pMJ601 (Sa/I, SmaI, Af/I, NarI, BspMII, BamHI, ApaI, NheI,SacII, KpnI, and HindIII cloning site; TK- and β-gal selection), andpTKgptF1S (EcoRI, PstI, Sa/I, AccI, HindII, SbaI, BamHI, and Hpa cloningsite, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toexpress the protein of the invention, a variant thereof, or an analog orderivative thereof. For example, the non-fusion pYES2 vector (XbaI,SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIIIcloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI,NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site,N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors that can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed. For example, expression ina bacterial system can be used to produce a nonglycosylated core proteinproduct.

Transgenic Animals

A host cell of the present invention also can be used to producenonhuman transgenic animals. For example, in one embodiment, a host cellof the invention is a fertilized oocyte or an embryonic stem cell intowhich sequences corresponding to SEQ ID NO:1 have been introduced. Suchhost cells then can be used to create non-human transgenic animals intowhich the exogenous sequences have been introduced into the genome, orhomologous recombinant animals in which endogenous sequences have beenaltered. Such animals are useful for studying the function and/oractivity of the protein of the invention and for identifying and/orevaluating modulators of the protein of the invention's activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in that one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians etc. A particular embodiment of the invention is aguinea pig that overexpresses the receptor of the invention and wouldhave utility as an animal model of allergic rhinitis, bronchial asthmaor chronic obstructive pulmonary disease.

As used herein, the term “transgene” refers to exogenous DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops and that remains in the genome of the mature animal. Thetransgene directs the expression of an encoded gene product in one ormore cell types or tissues of the transgenic animal. As used herein, a“homologous recombinant animal” is a non-human animal, preferably amammal, more preferably a mouse, in which an endogenous genecorresponding to SEQ ID. NO:1 has been altered by homologousrecombination. That is accomplished between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing anucleic acid molecule encoding SEQ ID NO:1 or a portion thereof into themale pronuclei of a fertilized oocyte using one of the transfectionmethods described above. The oocyte is then allowed to develop in apseudopregnant female foster animal. The cDNA sequence e.g., that of(SEQ ID NO: I), for example, can be introduced as a transgene into thegenome of a non-human animal. Alternatively, a nonhuman homologue of thehuman gene, such as a mouse gene, can be isolated based on hybridizationto the cDNA corresponding to SEQ ID NO:1, and used as a transgene.Intronic sequences and polyadenylation signals also can be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to thetransgene of the invention to direct expression of the protein of theinvention in particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, are conventional in the art and are described, for example, inU.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and inHogan, Manipulating the Mouse Embryo, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1986), the disclosures of each of whichare incorporated herein by reference. Similar methods are used forproduction of other transgenic animals with a transgene in the genomeand/or expression of mRNA of the invention in tissues or cells of theanimals. A transgenic founder animal then can be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding SEQ ID NO:1 can be bred further to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, a vector is prepared thatcontains at least a portion of the gene of the invention (e.g., a humanor a non-human homolog of the gene of the invention, e.g., a murinegene) into which a deletion, addition or substitution has beenintroduced thereby to alter, e.g., functionally disrupt, the gene of theinvention. In a particular embodiment, the vector is designed such that,on homologous recombination, the endogenous gene is disruptedfunctionally (i.e., no longer encodes a functional protein; alsoreferred to as a “knock out” vector).

Alternatively, the vector can be designed such that, on homologousrecombination, the endogenous gene is mutated or otherwise altered butstill encodes functional protein (e.g., an upstream regulatory regioncan be altered thereby altering the expression of the endogenousprotein).

In the homologous recombination vector, the altered portion of the geneis flanked at the 5′ and 3′ ends by an additional nucleic acid sequenceof the gene to allow for homologous recombination to occur between theexogenous gene carried by the vector and an endogenous gene in anembryonic stem cell. The additional flanking nucleic acid sequence is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see, e.g., Thomas etal., Cell (1987) 51:503 for a description of homologous recombinationvectors).

The vector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced gene of the inventionhas homologously recombined with the endogenous gene are selected (see,e.g., Li et al., Cell (1992) 69:915). The selected cells then areinjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed., IRL, Oxford,(1987) pp. 113-152). A chimeric embryo then can be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in the germcells can be used to breed animals in that all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene.

Methods for constructing homologous recombination vectors and homologousrecombinant animals are described further in Bradley, Current Opinion inBio/Technology (1991) 2:823-829 and in PCT Publication Nos. WO 90/11354,WO 91/01140, WO 92/0968 and WO 93/04169, the disclosures of which areincorporated by reference.

In another embodiment, transgenic non-human animals can be produced thatcontain selected systems to allow for regulated expression of thetransgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al., Proc Natl Acad Sci USA(1992) 89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al., Science (1991)251:1351-1355). If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe cre recombinase and a selected protein are required. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase.

Clones of the non-human transgenic animals described herein also can beproduced according to the methods described in Wilmut et al., Nature(1997) 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669(and which are hereby incorporated by reference herein in theirentireties). In brief, a cell, e.g., a somatic cell, from the transgenicanimal can be isolated and induced to exit the growth cycle and enter G₀phase. The quiescent cell then can be fused, e.g., through the use ofelectrical pulses, to an enucleated oocyte from an animal of the samespecies from which the quiescent cell is isolated. The reconstructedoocyte then is cultured such that it develops to morula or blastocyte,and then is transferred to a pseudopregnant female foster animal. Theoffspring borne of the female foster animal will be a clone of theanimal from that the cell, e.g., the somatic cell, is isolated.

Additional Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, antibodies ofthe present invention, and fragments of such moieties, may be used inone or more of the following methods: a) screening assays; b) detectionassays (e.g., chromosomal mapping, tissue typing, forensic biology); c)predictive medicine (e.g., diagnostic assays, prognostic assays,monitoring clinical trials and pharmacogenomics); and d) methods oftreatment (e.g., therapeutic and prophylactic). The protein of theinvention interacts with other cellular proteins, and thus can be usedfor (i) regulation of cellular proliferation; (ii) regulation ofcellular differentiation; (iii) regulation of cell survival, and (iv)regulation of cell function. The isolated nucleic acid molecules of theinvention can be used to express the protein of the invention (e.g., viaa recombinant expression vector in a host cell in gene therapyapplications), to detect mRNA of the invention (e.g., in a biologicalsample) or to detect a genetic lesion in a gene of the invention and tomodulate activity of endogenous mRNA, DNA or protein. In addition, aprotein of the invention can be used to screen drugs or compounds thatmodulate the protein activity or expression, as well as to treatdisorders characterized by insufficient or excessive production ofendogenous protein. Screening for the production of protein forms thathave decreased or aberrant activity compared to wild type protein canalso be performed with the present invention. In addition, an antibodyof the invention can be used to detect and to isolate proteins and tomodulate protein activity. The invention further pertains to novelagents identified by the above-described screening assays and usesthereof for treatments as described herein.

1. Detection and Screening Assays

Activation of a G protein receptor in the presence of endogenous ligandallows for G protein receptor complex formation, thereupon leading tothe binding of GTP to the G protein. The GTPase domain of the G proteinslowly hydrolyzes the GTP to GDP resulting, under normal conditions, inreceptor deactivation. However, constitutively activated receptorscontinue to hydrolyze GDP to GTP.

A non-hydrolyzable substrate of G protein, [³⁵S]GTPγS, can be used tomonitor enhanced binding to membranes which express constitutivelyactivated receptors. Traynor and Nahorski reported that [³⁵S]GTPγS canbe used to monitor G protein coupling to membranes in the absence andpresence of ligand (Traynor et al., Mol Pharmacol (1995) 47(4):848-54).A preferred use of such an assay system is for initial screening ofcandidate compounds, since the system is generically applicable to all Gprotein-coupled receptors without regard to the particular G proteinthat binds to the receptor.

G_(s) stimulates the enzyme adenylyl cyclase, while G_(i) and G_(o)inhibit that enzyme. As is well known in the art, adenylyl cyclasecatalyzes the conversion of ATP to cAMP; thus, constitutively activatedGPCRs that couple the G_(s) protein are associated with increasedcellular levels of cAMP. Alternatively, constitutively activated GCPRsthat might couple the G_(i) (or G_(o)) protein are associated withdecreased cellular levels of cAMP. See “Indirect Mechanism of SynapticTransmission”, Chpt.8, from Neuron to Brain (3^(rd) Ed.), Nichols et al.eds., Sinauer Associates, Inc., 1992. Thus, assays that detect cAMP canbe used to determine if a candidate compound is an inverse agonist tothe receptor. A variety of approaches known in the art for measuringcAMP can be utilized. In one embodiment, anti-cAMP antibodies are usedin an ELISA-based format. In another embodiment, a whole cell secondmessenger reporter system assay is contemplated (see PCT Publication No.WO 00/22131 and incorporated by reference herein in their entireties). Aparticular embodiment is the SPA assay described below in “Example 5”.

In a related aspect, cyclic AMP drives gene expression by promoting thebinding of a cAMP-responsive DNA binding protein or transcription factor(CREB) which then binds to the promoter at specific sites called cAMPresponse elements, and drives the expression of the gene. Thus, reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Further, as a constitutively activated G_(s)-linkedreceptor causes the accumulation of cAMP, that then activates the geneand expression of the reporter protein. The reporter protein, such asβ-galactosidase or luciferase, then can be detected using standardbiochemical assays (PCT Publication No. WO 00/22131 incorporated byreference herein).

Other G proteins, such as G_(o) and G_(q), are associated withactivation of the enzyme, phospholipase C, which in turn hydrolyzes thephospholipid, PIP2, releasing two intracellular messengers:diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Increasedaccumulation of IP3 is associated with activation of G_(q)-associatedreceptors and G_(o)-associated receptors (PCT Publication No. WO00/22131 incorporated by reference herein). Assays that detect IP3accumulation can be used to determine if a candidate compound is aninverse agonist to a G_(q)-associated receptor or a G_(o)-associatedreceptor. G_(q)-associated receptors also can be examined using an AP1reporter assays that measures whether G_(q)-dependent phospholipase Ccauses activation of genes containing AP1 elements. Thus, activatedG_(q)-associated receptors will demonstrate an increase in theexpression of such genes, whereby inverse agonists will demonstrate adecrease in such expression.

Also provided herein is a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules orother drugs) that bind to proteins of the invention or have astimulatory or inhibitory effect on, for example, expression or activityof the protein. For example, the screening assays described herein couldbe used to identify compounds acting as antagonists at the receptor thatwould have utility for treating bronchial asthma.

In one embodiment, the invention provides assays for screening candidateor test compounds that bind to or modulate the activity of themembrane-bound form of the protein of the invention, polypeptide orbiologically active portion thereof. The test compounds of the presentinvention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, Anticancer Drug Des (1997) 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc Natl Acad Sci USA(1993) 90:6909; Erb et al., Proc Natl Acad Sci USA (1994) 91:11422;Zuckermann et al., J Med Chem (1994) 37:2678; Cho et al., Science (1993)261:1303; Carrell et al., Angew Chem Int Ed Engl (1994) 33:2059; Carellet al., Angew Chem Int Ed Engl (1994) 33:2061; and Gallop et al., J MedChem (1994) 37:1233.

Libraries of compounds may be presented in solution (e.g., HoughtenBio/Techniques (1992) 13:412-421) or on beads (Lam, Nature (1991)354:82-84), chips (Fodor, Nature (1993) 364:555-556), bacteria (U.S.Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA (1992)89:1865-1869) or phage (Scott et al., Science (1990) 249:386-390;Devlin, Science (1990) 249:404-406; Cwirla et al., Proc Natl Acad SciUSA (1990) 87:6378-6382; and Felici, J Mol Biol (1991) 222:301-310); thedisclosures of each of the foregoing references are incorporated hereinby reference

In a particular embodiment of the present invention, an assay is acell-based assay in which a cell that expresses a membrane-bound form ofthe protein of the invention, or a biologically active portion thereof,on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to the protein is determined. The cell, forexample, can be a yeast cell or a cell of mammalian origin. Determiningthe ability of the test compound to bind to the protein can beaccomplished, for example, by coupling the test compound with aradioisotope or enzymatic label so that binding of the test compound tothe protein of the invention or biologically active portion thereof canbe determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C or ³H, eitherdirectly or indirectly and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Alternatively, testcompounds can be labeled enzymatically with, for example, horseradishperoxidase, alkaline phosphatase or luciferase and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct. In a particular embodiment, the assay comprises contacting acell that expresses a membrane-bound form of the protein of theinvention or a biologically active portion thereof, on the cell surfacewith a known compound that binds the protein to form an assay mixture.Then, contacting the assay mixture with a test compound and determiningthe ability of the test compound to interact with the protein, whereindetermining the ability of the test compound to interact with theprotein comprises determining the ability of the test compound to bindpreferentially to the protein of the invention or a biologically activeportion thereof as compared to the known compound.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a membrane-bound form of the protein of theinvention or a biologically active portion thereof, on the cell surfacewith a test compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of the protein of the invention or abiologically active portion thereof can be accomplished, for example, bydetermining the ability of the protein to bind to or to interact with atarget molecule. As used herein, a “target molecule” is a molecule withwhich the protein of the invention binds or interacts with in nature,for example, a molecule on the surface of a cell that expresses theprotein of the invention, a molecule on the surface of a second cell, amolecule in the extracellular milieu, a molecule associated with theinternal surface of a cell membrane or a cytoplasmic molecule. A targetmolecule can be another molecule or a protein or polypeptide of thepresent invention. In one embodiment, a target molecule is a componentof a signal transduction pathway that facilitates transduction of anextracellular signal (e.g., a signal generated by binding of a compoundto a membrane-bound protein of the invention) through the cell membraneand into the cell. The target, for example, can be a secondintercellular protein that has catalytic activity or a protein thatfacilitates the association of downstream signaling molecules.

Determining the ability of the protein of the instant application tointeract with a target molecule can be accomplished by one of themethods described above for determining direct binding. In a particularembodiment, determining the ability of the protein of the invention tobind to or to interact with a target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target (e.g., intracellular Ca²⁺,diacylglycerol, IP3 etc.), detecting catalytic/enzymatic activity of thetarget on an appropriate substrate, detecting the induction of areporter gene (e.g., a responsive regulatory element operably linked toa nucleic acid encoding a detectable marker, e.g. luciferase) ordetecting a cellular response, e.g., cellular differentiation,proliferation or function. A particular embodiment is described below in“Example 4” where the receptor of the invention is coupled to Gα16 toelicit a calcium response.

The present invention further extends to a cell-free assay comprisingcontacting a protein of the invention, or biologically active portionthereof, with a test compound, and determining the ability of the testcompound to bind to the protein or biologically active portion thereof.Binding of the test compound to the protein can be determined eitherdirectly or indirectly as described above. In a preferred embodiment,the assay includes contacting the protein of the invention orbiologically active portion thereof with a known compound that binds theprotein to form an assay mixture. Then, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with the protein. Wherein, determining the ability of thetest compound to interact with the protein of the invention comprisesdetermining the ability of the test compound to preferentially bind tothe protein or biologically active portion thereof as compared to theknown compound.

Another cell-free assay of the present invention involves contacting theprotein of the invention or biologically active portion thereof, with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of the protein can be accomplished,for example, by determining the ability of the protein to bind to atarget molecule by one of the methods described above for determiningdirect binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of the protein can beaccomplished by determining the ability of the protein to furthermodulate a target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as described previously.

Still another cell-free assay of the present invention comprisescontacting the protein of the invention or biologically active portionthereof, with a known compound that binds the protein to form an assaymixture, contacting the assay mixture with a test compound anddetermining the ability of the test compound to interact with theprotein. The step for determining the ability of the test compound tointeract with the protein comprises determining the ability of theprotein preferentially to bind to or to modulate the activity of atarget molecule.

Receptors can be activated by non-ligand molecules that necessarily donot inhibit ligand binding but cause structural changes in the receptorto enable G protein binding or, perhaps receptor aggregation,dimerization or clustering that can cause activation. For example,antibodies can be raised to the various portions of the receptor of theinvention that are exposed at the cell surface. Those antibodiesactivate a cell via the G protein cascade as determined by standardassays, such as monitoring cAMP levels or intracellular Ca⁺² levels.Because molecular mapping, and particularly epitope mapping, isinvolved, monoclonal antibodies may be preferred. The monoclonalantibodies can be raised both to intact receptor expressed at the cellsurface and peptides known to form at the cell surface. The method ofGeysen et al., U.S. Pat. No. 5,998,577, can be practiced to obtain aplurality of relevant peptides.

Antibodies found to activate the receptor of the invention may bemodified to minimize activities extraneous to receptor activation, suchas complement fixation. Thus, the antibody molecules can be truncated ormutated to minimize or to remove activities outside of receptoractivation. For example, for certain antibodies, only theantigen-binding portion is needed. Thus, the F_(c) portion of theantibody can be removed.

Cells expressing the receptor of the invention are exposed to antibodyto activate the receptor. Activated cells then are exposed to variousmolecules in order to identify which molecules modulate receptoractivity, and result in higher activation levels or lower activationlevels. Molecules that achieve those goals then can be tested on cellsexpressing the receptor of the invention without antibody to observe theeffect on non-activated cells. The target molecules then can be testedand modified as candidate drugs for the treatment of disordersassociated with altered metabolism using known techniques.

The cell-free assays of the present invention are amenable to use ofboth the soluble form and the membrane-bound form of the protein of theinvention. In the case of cell-free assays comprising the membrane-boundform, it may be desirable to utilize a solubilizing agent such that themembrane-bound form is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON X-100,TRITON X-114, THESIT, isotridecylpoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propane sulfonate(CHAPSO) or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either the protein of theinvention or a target molecule thereof to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a test compound tothe protein of the invention or interaction of the protein with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtitre plates, test tubes andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided that adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/protein ofthe invention fusion proteins or glutathione-S-transferase/target fusionproteins can be adsorbed onto glutathione SEPHAROSE beads (SigmaChemical, St. Louis, Mo.). Alternatively, glutathione-derivatizedmicrotitre plates are then combined with the test compound.Subsequently, either the non-adsorbed target protein or the protein ofthe invention and the mixture are incubated under conditions conduciveto complex formation (e.g., at physiological conditions for salt andpH). Following incubation, the beads or microtitre plate wells arewashed to remove any unbound components, and the presence of complexformation is measured either directly or indirectly. Alternatively, thecomplexes can be dissociated from the matrix and the level of binding oractivity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theprotein of the invention or a target molecule thereof can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated proteinof the invention or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized inthe wells of streptavidin-coated 96-well plates (Pierce Chemicals).Alternatively, antibodies that are reactive with proteins of theinvention or a target molecule, but do not interfere with binding of theprotein of the invention to the target molecule, can be derivatized tothe wells of the plate. Upon incubation, unbound target or protein ofthe invention can be trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with proteins of the invention ortarget molecule, as well as enzyme-linked assays that rely on detectingan enzymatic activity associated with the protein of the invention ortarget molecule.

In another embodiment, modulators of protein expression are identifiedin a method wherein a cell is contacted with a candidate compound, andthe expression of mRNA or protein of the invention in the cell isdetermined. The level of expression of mRNA or protein in the presenceof the candidate compound is compared to the level of expression of mRNAor protein in the absence of the candidate compound. The candidatecompound then can be identified as a modulator of expression based onthat comparison. For example, when expression of mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in the absence thereof, the candidate compoundis identified as a stimulator or agonist of mRNA or protein expression.Alternatively, when expression of mRNA or protein is less (statisticallysignificantly less) in the presence of the candidate compound than inthe absence thereof, the candidate compound is identified as aninhibitor or antagonist of mRNA or protein expression. If activity isreduced in the presence of ligand or agonist, or in a constitutivelyexpressing cell is below baseline, the candidate compound is identifiedas an inverse agonist. The level of mRNA or protein expression in thecells can be determined by methods described herein for detecting mRNAor protein.

In yet another aspect of the invention, the proteins of the inventioncan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell (1993)72:223-232; Madura et al., J Biol Chem (1993) 268:12046-12054; Bartel etal., Bio/Techniques (1993) 14:920-924; Iwabuchi et al., Oncogene (1993)8:1693-1696; and PCT Publication No. WO 94/10300, the disclosures ofeach of which are incorporated herein by reference), to identify otherproteins that bind to or interact with the protein of the invention andmodulate the activity of the protein of the invention. Such bindingproteins are also likely to be involved in the propagation of signals bythe proteins of the invention such as, upstream or downstream elementsof the signaling pathway.

Since the present invention enables the production of large quantitiesof pure protein of the instant application, physical characterization ofthe conformation of areas of likely function can be ascertained forrational drug design. For example, the intracellular and extracellulardomains are regions of particular interest. Once the shape and ionicconfiguration of a region is discerned, candidate drugs that shouldinteract with those regions can be configured and then tested in intactcells, animals and patients. Methods that would enable deriving such 3-Dstructure information include X-ray crystallography, NMR spectroscopy,molecular modeling and so on. The 3-D structure also can lead toidentification of analogous conformational sites in other known proteinswhere known drugs that interact at this site exist. These drugs, orderivatives thereof, may find use with protein of the present invention.

The screening assays described above would be of particular utility inidentifying compounds acting as an agonist, partial agonist, antagonist,inverse agonist or modulator of the receptor of the invention providinga means to identify compounds for the treatment of disease including,but not limited to, bronchial asthma, COPD, allergic rhinitis, allergicdermatitis, allergic conjuctivitis, systemic mastocytosis and ischemicreperfusion injury.

The invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

Portions or fragments of the DNA sequences of the present invention canbe used in numerous ways as polynucleotide reagents. For example, thesequences can be used to: (i) map the respective genes on a chromosomeand, thus, locate gene regions associated with genetic disease; (ii)identify an individual from a minute biological sample (tissue typing);and (iii) aid in forensic identification of a biological sample. Theapplications are described in the subsections below.

2. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, the sequence can be used to map the location of the gene ofthe present invention on a chromosome. Accordingly, nucleic acidmolecules described herein or fragments thereof can been used to map thelocation in a genome. The mapping of the location of the sequence in agenome, particularly a human genome, is an important first step incorrelating the sequences with genes associated with disease.

Briefly, genes can be mapped in a genome by preparing PCR primers(preferably 15-25 bp in length) from the sequences disclosed in SEQ IDNO:1. The primers are used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to sequences of the invention yield anamplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, generally human chromosomes are lost in randomorder, but the mouse chromosomes are retained. By using media in whichmouse cells cannot grow (because of lack of a particular enzyme), but inwhich human cells can grow, the one human chromosome that contains thegene encoding the needed enzyme will be retained. By using variousmedia, panels of hybrid cell lines are established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes. (D'Eustachioet al., Science (1983) 220:919-924). Somatic cell hybrids containingonly fragments of human chromosomes also can be produced by using humanchromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermocycler.

Other mapping strategies that can similarly be used to map a sequence toa particular chromosome in a genome include in situ hybridization(described in Fan et al., Proc Natl Acad Sci USA (1990) 87:6223-27),pre-screening with labeled flow-sorted chromosomes and pre-selection byhybridization to chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can also be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells in which division has been blocked in metaphase by a chemical,e.g., colcemid, that disrupts the mitotic spindle. The chromosomes canbe treated briefly with trypsin and then stained with Giemsa. A patternof light and dark bands develops on each chromosome so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases and more preferably, 2,000 bases willsuffice to get good results in a reasonable amount of time. For a reviewof the technique, see Verma et al. (Human Chromosomes: A Manual of BasicTechniques (Pergamon Press, New York, 1988)). Chromosomal mapping can beinferred in silico, and employing statistical considerations, such aslod scores or mere proximity.

Reagents for chromosome mapping can be used individually to locate asingle site on a chromosome. Furthermore, panels of reagents can be usedfor marking multiple sites and/or multiple chromosomes. Reagentscorresponding to flanking regions of the gene actually are preferred formapping purposes. Coding sequences are more likely to be conservedwithin gene families, thus increasing the chance of cross hybridizationduring chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity, Welch Medical Library). The relationship between genes anddisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, e.g., Egeland et al., Nature (1987) 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the protein of theinvention can be determined. If a mutation is observed in some or all ofthe affected individuals, but not in any unaffected individuals, thenthe mutation is likely to be the causative agent of the particulardisease. Comparison of affected and unaffected individuals generallyinvolves first looking for structural alterations in the chromosomessuch as deletions or translocations that are visible from chromosomespreads or detectable using PCR based on that DNA sequence. Ultimately,complete sequencing of genes from several individuals can be performedto confirm the presence of a mutation and to distinguish mutations frompolymorphisms.

3. Diagnostic Assays

An exemplary method for detecting the presence or absence of a nucleicacid or protein of the invention in a biological sample involvesobtaining a biological sample from a test subject and contacting thebiological sample with a compound or an agent capable of detecting theprotein or nucleic acid (e.g., mRNA or genomic DNA) such that thepresence is detected in the biological sample. A preferred agent fordetecting mRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to the mRNA or genomic DNA of the invention. The nucleicacid probe can be, for example, a full-length nucleic acid, such as thenucleic acid of SEQ ID NO:1 or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 or morenucleotides in length and sufficient to specifically hybridize understringent conditions to mRNA or genomic DNA. Other suitable probes foruse in the diagnostic assays of the invention are described herein.

A particular agent for detecting the protein of the invention is anantibody capable of binding to the protein, preferably an antibody witha detectable label. Antibodies can be polyclonal, chimeric, or morepreferably, monoclonal. An intact antibody or a fragment thereof (e.g.,F_(ab) or F_((ab′)2)) can be used. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.That is, the detection method of the invention can be used to detectmRNA, protein or genomic DNA in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of mRNA includeNorthern hybridization and in situ hybridization. In vitro techniquesfor detection of the protein include ELISA, Western blot,immunoprecipitation and immunofluorescence. In vitro techniques fordetection of genomic DNA include Southern hybridization. Furthermore, invivo techniques for detection of protein include introducing into asubject a labeled antibody against the protein of the invention. Forexample, the antibody can be labeled with a radioactive marker, thepresence and location of which in a subject can be detected by standardimaging techniques.

In an embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A particular biological sample having applications herein is aneutrophil sample isolated by conventional means from a subject.

Hence, association with a disease and identification of nucleic acid orprotein polymorphism diagnostic for the carrier or the affected can bebeneficial in developing prognostic or diagnostic assays. For example,it would be beneficial to have a prognostic or diagnostic assay forrheumatoid arthritis, asthma, Crohn's Disease and so on. Expression ofthe nucleic acid or protein of the invention is elevated in cellsassociated with activated or inflammatory states. Disorders associatedwith inflammation include, anaphylactic states, colitis, Crohn'sDisease, edematous states, contact hypersensitivity, allergy, otherforms of arthritis, meningitis and other conditions wherein the immunesystem reacts to an insult by vascular dilation, heat, collecting cells,fluids and the like at a site resulting in swelling and the like. Thus,a disorder in metabolism may be diagnostic for rheumatoid arthritis.Moreover, the molecular mechanism of rheumatoid arthritis may bedetectable, such as, there may be a diagnostic SNP, RFLP, variability ofexpression level, variability of function and so on, that can bedetectable in a tissue sample, such as a blood sample.

In another embodiment, the methods further involve obtaining abiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting protein, mRNA or genomicDNA of the invention, such that the presence and amount of protein, mRNAor genomic DNA is detected in the biological sample, and then comparingthe presence and amount of protein, mRNA or genomic DNA in the controlsample with the presence and amount of protein, mRNA or genomic DNA in atest sample.

4. High Throughput Assays of Chemical Libraries

Any of the assays for compounds capable of modulating the activity ofnucleic acid or protein of the invention are amenable to high throughputscreening. High throughput screening systems are commercially available(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries,Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; PrecisionSystems, Inc., Natick, Mass., etc.). These systems typically automateentire procedures including all sample and reagent pipetting, liquiddispensing, timed incubations, and final readings of the microplate indetector(s) appropriate for the assay. These configurable systemsprovide high throughput and rapid start up as well as a high degree offlexibility and customization. The manufacturers of such systems providedetailed protocols of the various high throughput protocols. Thus, forexample, Zymark Corp. provides technical bulletins describing screeningsystems for detecting the modulation of gene transcription, ligandbinding, and the like.

5. Kits

The invention also encompasses kits for detecting the presence of thenucleic acid or protein of the invention in a biological sample (a testsample). Such kits can be used to determine if a subject is sufferingfrom or is at increased risk of developing a disorder associated withaberrant expression (e.g., an immunological disorder). For example, thekit can comprise a labeled compound or agent capable of detectingprotein or mRNA of the invention in a biological sample and means fordetermining the amount of nucleic acid or protein in the sample (e.g.,an antibody or an oligonucleotide probe). Kits also can be used to yieldresults indicating whether the tested subject is suffering from or is atrisk of developing a disorder associated with aberrant expression ofnucleic acid or protein of the invention, if the amount of protein ormRNA is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to the proteinof the invention; and, optionally, (2) a second, different antibody thatbinds to the protein of the invention or to the first antibody and isconjugated to a detectable agent. If the second antibody is not present,then either the first antibody can be detectably labeled, oralternatively, another molecule that binds the first antibody can bedetectably labeled. In any event, a labeled binding moiety is includedto serve as the detectable reporter molecule, as known in the art.

For oligonucleotide-based kits, a kit of the present invention cancomprise, for example: (1) an oligonucleotide, e.g., adetectably-labeled oligonucleotide, that hybridizes to a nucleic acidsequence of the invention or (2) a pair of primers useful for amplifyinga nucleic acid molecule of the invention.

The kit also can comprise, e.g., a buffering agent, a preservative or aprotein stabilizing agent. The kit also can comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). Furthermore, the kit may also contain a control sample orseries of control samples that can be assayed and compared to the testsample. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package. Instructions and background information may also beenclosed.

6. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs or compounds) on theexpression or activity of the nucleic acids or proteins of the invention(e.g., the ability to modulate aberrant cell proliferation,differentiation and/or function) can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent, as determined by a screening assay as described herein, toincrease gene expression, protein levels or protein activity, can bemonitored in clinical trials of subjects exhibiting decreased geneexpression, protein levels or protein activity. Alternatively, theeffectiveness of an agent, as determined by a screening assay, todecrease gene expression, protein levels or protein activity, can bemonitored in clinical trials of subjects exhibiting increased geneexpression, protein levels or protein activity. In such clinical trials,expression or activity and preferably, that of other genes that havebeen implicated in, for example, a cellular proliferation disorder, canbe used as a marker of the immune responsiveness of a particular cell.For example, and not by way of limitation, genes, including the genes ofthe invention, that are modulated in cells by treatment with an agent(e.g., compound, drug or small molecule) that modulates activity of thenucleic acid or protein of the invention (e.g., as identified in ascreening assay described herein) can be identified. Thus, to study theeffect of agents on cellular proliferation disorders, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe levels of expression of nucleic acids of the invention and othergenes implicated in the disorder. The levels of gene expression (i.e., agene expression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced by one of the methods as described herein or bymeasuring the levels of activity of genes of the invention or othergenes. In that way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, the response state may be determined before and at variouspoints during treatment of the individual with the agent.

In a particular embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule or other drug candidate identified by the screeningassays described herein) comprising the steps of (i) obtaining apre-administration sample from a subject prior to administration of theagent; (ii) detecting the level of expression of a protein, mRNA orgenomic DNA of the invention in the preadministration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the protein, mRNA orgenomic DNA of the invention in the post-administration samples; (v)comparing the level of expression or activity of the protein, mRNA orgenomic DNA of the invention in the pre-administration sample with theprotein, mRNA or genomic DNA in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of the protein,mRNA or genomic DNA of the invention to higher levels than detected,i.e., to increase the effectiveness of the agent. Alternatively,decreased administration of the agent may be desirable to decreaseexpression or activity of the protein, mRNA or genomic DNA of theinvention to lower levels than detected, i.e., to decrease theeffectiveness of the agent.

The following examples describe the invention in greater detail.

EXAMPLES Example 1

Cloning of an Initial Exonic DNA Fragment of the Guinea Pig DP Receptor

Cloning of the Cavia porcellus DP receptor cDNA was initiated by cloningan exonic fragment of the DP receptor from genomic DNA using PCR. Arange of PCR primers were designed using the conserved regions of theHuman (U31332), Mouse (NM_(—)008962) and Rat (NM_(—)022241) DP receptorsequences that aligned using the program Sequencher (Gene Codes, AnnArbor Mich.). Cavia porcellus (guinea pig) genomic DNA was purchasedfrom CeMines (Evergreen, Co.). The primer used to amplify a 420 bpfragment of Cavia genomic DNA was performed with the primers 675_Topo_F3(SEQ ID NO: 3: GGGACACCCTTTCTTCTACAA) and 675_Topo_R2 (SEQ ID NO: 4:GAACACATGGTGAAGAGCACTG). The PCR product was cloned using TOPO-TAcloning (Invitrogen, Carlsbad Calif.) and the insert was sequenced on anABI 3100 DNA sequencer according to the manufacturer's instructions.

3′ and 5′ RACE-PCR Cloning

The resulting DNA sequence was aligned to the human, mouse and rat DPsequences. The alignment revealed that the PCR product sequence washomologous to, but yet distinct from, the DP receptor consensus from thespecies examined (FIG. 3). The Cavia porcellus DP receptor consensus wasused to design additional primers to extend the cloned sequence usingthe rapid amplification of DNA ends (RACE) method. In order to obtain 3′end of the DP receptor transcript, the SMART RACE system from Clontech(a subsidiary of BD Biosciences, Palo Alto Calif.) was employed. Theprimer 675_GP_(—)3′RACE_F (SEQ ID NO: 5: GTGCTCGTGGCGCCGGTGTG) was usedwith Cavia lung mRNA converted to a cDNA template to extend the Cavia DPreceptor mRNA sequence. RACE products were cloned into PCR4-Topo(Invitrogen, Carlsbad Calif.), sequenced and aligned as described aboveto reveal complete 3′ extension of the coding sequence of the DPreceptor. In order to isolate the 5′ end of the Cavia DP receptor cDNA,SMART RACE was performed with the primer 675_Rev_P2 (SEQ ID NO: 6:CACATGGTGAAGAGCACGGTCATGA) and a 1 kb PCR product was generated. Thepurified PCR product was used as a template for 5′ Nested RACE using theprimer 675_RACE_R9 (SEQ ID NO: 7: TCACCAGGCACTTGCCTAGCAGGTCTGT). TheRACE products were cloned into PCR4-Topo (Invitrogen), sequenced andaligned as described above to reveal complete 5′ extension of the codingsequence of the DP receptor.

Construction of cDNA Encoding the Guinea Pig DP Receptor

The coding sequence of the DP receptor was identified using the programGene Construction Kit (Textco, Keene N.H.). Gateway cloning compatibleprimers were designed to flank the coding sequence (GW675, forwardprimer SEQ ID NO: 8: AAAAGCAGGCTTAGGAATGTCCTTCTATCCCTGCAACAC; GW675,reverse primer SEQ ID NO: 9 AAGAAAGCTGGGTCTCACAGACTGGATTCCACGTTAG), andutilized in a PCR reaction with cDNA generated from the Cavia porcellusovalbumin-stimulated lung cells. PCR was performed using 10 units of PFUTurbo (Stratagene, La Jolla Calif.) thermostable polymerase and 100 ngof template cDNA. A 1.1 kb DNA fragment was generated, purified by gelelectrophoresis chromatography by the QiaQuick protocol (Qiagen) andcloned into the pDONR201 vector using the Gateway BP recombinase cloningmethod (Invitrogen). Cloning reactions were transformed into E. coliDH5-alpha and mini-prep DNA from the resultant colonies were subjectedto DNA sequencing to confirm cloning of the complete DP receptor codingsequence.

Example 2

Northern Blot Analysis

Northern blot analysis was performed with the initial genomic DNAfragment of the Cavia DP receptor (FIG. 5). Lung was isolated from maleHartley Guinea pigs that had been and challenged with ovalbumin or hadnot received ovalbumin treatment. Total lung RNA expression fromunchallenged Cavia porcellus (Lane 2) was compared to total lung RNAexpression from ovalbumin challenged Cavia porcellus (Lane 3). RNAloaded was equivalent as determined by spectroscopy and intensity of the18S RNA band. Northern blotting for the DP receptor identified a 3-4 kbmRNA in the guinea pig lung, a size consistent with the transcriptreported for mouse and human DP (Hirata et al., 1994; Boie et al.,1995). This mRNA was significantly upregulated in the lungs of guineapigs that had been sensitized and challenged with ovalbumin a resultcomparable with that reported previously for DP receptor beingupregulated in the mouse lung on antigen challenge (Matsuoka et al.,2000). These data support the importance of DP in the asthmatic responsein the guinea pig lung.

Example 3

Sequence Analysis of Guinea Pig to Orthologue DP Receptors

The nucleotide sequence (FIG. 1) and deduced amino acid sequence (FIG.2) for the guinea pig DP receptor is shown. The guinea pig DP cDNAcontains a 1,032 bp open reading frame which encodes a 345 amino acidprotein with a calculated molecular mass of 38,250.

The guinea pig DP protein contains two potential N-glycosylation sites,Asn-7 in the amino terminus and Asn-86 in the first extracellular loop.There are also 2 potential protein kinase C phosphorylation sites,Ser-46 and Thr-140 located in the first and third cytoplasmic loops,respectively.

The nucleotide sequence of the guinea pig DP receptor compared with thecorresponding sequences of human, rat and mouse DP are shown in FIG. 1.Similarly at the protein level, the sequence identity against the guineapig DP receptor was 66% for human DP, 63% for mouse DP and 65% for ratDP (FIG. 2).

Hydropathy analysis confirmed the presence of seven putativetransmembrane domains which mapped identically to conserved areas thathad previously been defined in the sequences of mouse, rat and human DP.Sequence conservation was the highest in the transmembrane domainsbetween the DP orthologues. Two sequence stretches that had previouslybeen reported to be characteristically conserved amongst GPCRs of theprostanoid family (Hirata et al., 1994) were also present in the guineapig DP protein: QYCPGTWCR (SEQ ID NO: 10) in the second extracellularloop and RFLSVISIVDPWIFI (SEQ ID NO: 11) in the seventh transmembranedomain were identical among all DP orthologues.

The extracellular loop between TMDs VI and VII also showed differencesbetween species. This loop varied between the orthologues and hadlengths of 24, 21, 21 and 18 amino acids in human, rat, mouse and guineapig DP, respectively. Of particular note is the loss of 6 amino acids inguinea pig DP between TMDs VI and VII compared to the human DP receptor.Furthermore, 3 amino acids are also removed in this region in both themouse and rat DP receptors. Kobayashi et al (2000) generated a series ofchimeric IP-DP receptors to define the regions that confer the ligandbinding selectivity of DP. It is interesting to note that one of theregions they concluded to be important in selective and potent bindingof PGD2 was the transmembrane VI-VII region, the exact same region shownto be 6 amino acids shorter in this newly cloned guinea pig DP receptor.Orthologue differences in ligand affinity or compound potency may be dueto interactions within the TMD VI-VII loop and the alterations in thisloop on the guinea pig receptor.

The first and third intracellular loops are 3 and 5 amino acids shorterin the guinea pig DP protein, whereas in the mouse, human and rat DPproteins these intracellular loops are all of identical size. Kobayashiet al. also highlighted the importance of the transmembrane domain 1 tothe first extracellular loop region for PGD2 binding. Since the firstintracellular loop is 3 amino acids shorter in guinea pig DP compared tohuman, mouse or rat DP, this region could be an additional regioncontributing to receptor binding affinity. Additionally, this region ofthe receptor could attribute to the differences observed between theaffinities of compounds to human and guinea pig DP. The thirdintracellular loop (between TMDs V and VI) is 5 amino acids shorter inguinea pig DP, providing another region on the receptor contributing tofunctional relevance of PGD2.

Example 4

Construction of pEAK10-gpDP and pEAK10-mDP Mammalian Expression Vectors

A full length cDNA for the mouse DP receptor was obtained by PCR andcloned into the pDONR201 vector using the Gateway BP recombinase cloningmethod. This generated a mouse DP vector that was analogous to theguinea pig DP vector described above. DNA sequencing confirmed that thismouse DP cDNA was identical to the previously described mouse DPsequence defined by Genbank accession number NM_(—)008962. Forexpression studies, the mouse and guinea pig DP receptors were subclonedby an LR reaction into a pEAK10 expression vector (Edge Biosystems) thathad been previously gateway adapted. Gateway adaptation of the pEAK10vector was conducted by digesting with EcoRI and subsequent Klenowfilling for cloning of the Gateway cassette into the vector. Theresultant vectors pEAK10-gpDP and pEAK10-mDP were used for generation ofstable cell lines as described below.

Generation of a HEK293-Gα16 Cell Line

The cDNA encoding human Gα16 was cloned as described (Amatruda et al.,1991). Briefly, total RNA from HL-60 human promyelocytic leukemia cellswas isolated and used as a template for PCR-mediated synthesis of cDNAencoding Gα16. The resulting PCR product was cloned into the expressionvector pHook-3 (Invitrogen), which also coexpresses a single-chainantibody (sFv) to allow convenient enrichment of transfected cells usinga panning protocol with hapten-coated magnetobeads (Chesnut et al.,1996). HEK293 cells were transfected with the constructed plasmid(pGα16), selected with Zeocin and positive clones enriched usingmagnetobeads according to protocols supplied by the vendor. For finalpurification and selection, single clones were grown individually andassayed for functional expression of Gα16 by additional transfection ofan aliquot with an expression vector for an arbitrarily chosen GPCRnaturally coupling to Gαs (GIP receptor), with subsequent testing oftransfected cells for calcium signalling using the FLIPR device fromMolecular Devices Corp.

Expression of pEAK10-gpDP and pEAK10-mDP in HEK293-Gα16 Cells.

The pEAK10-gpDP and pEAK10-mDP vectors were transfected into theHEK293-Gα16 cell line using Lipofectamine 2000 (Gibco) as described bythe manufacturer. Transfected cells were cultured under selection with 1ug/ml puromycin and 250 ug/ml zeocin for 5 weeks. Expression of the DPreceptor was monitored in the transfected cell population by measuringthe release of intracellular calcium in response to PGD2 stimulation.

Intracellular Calcium Assays

For functional characterization the newly cloned guinea pig DP receptorwas stably transfected into HEK293-Gα16 cells and for comparison anequivalent cell line was generated with the mouse DP receptor. Both DPreceptor-expressing cell lines, as well as the parental cell line notexpressing any transfected DP receptor, were evaluated in a secondmessenger assay using the force coupling of the receptor to Gα16 toelicit a calcium response. Intracellular calcium measurements wereperformed using non-transfected HEK293-Gα16 cells or cells transfectedwith either the pEAK10-gpDP or the pEAK10-mDP expression vectors.Transfected and non-transfected cells were plated in 384 well plates at10,000 cells per well. Cells were washed three times with calcium assaybuffer. Cells were then incubated with 4 μM of the calcium loading dyeFura-4/AM (Molecular Probes) at 37° C. for x min. Unincorporatedfura-4/AM was removed by three further washes with calcium assay buffer.Intracellular calcium was measured following PGD2 or buffer stimulationof Fura-4/AM loaded cells using a FLIPR instrument (Molecular DevicesCorp.). As shown in FIG. 6, PGD2 stimulation caused robust increases inintracellular calcium mobilization in both the guinea pig and mouseDP-expressing cell lines with EC50 values of 1.4 nM and 18 nM,respectively. The maximum calcium release in both cell lines wascomparable. In contrast the parental HEK293-Gα16 cell line only showed acalcium response at very high PGD2 concentrations (i.e., above 10 μMPGD2).

Example 5

SPA cAMP Assay

An additional functional characterization of the newly cloned guinea pigDP receptor used the natural signaling pathway for DP, the stimulationof cAMP production by adenylate cyclase. Transfected or non-transfectedcells were plated at 40,000 cells per well of a 96 well plate. Afterovernight incubation at 37° C., medium was replaced and cells werestimulated with defined concentrations of PGD2 for 15 minutes. Theaccumulation of cAMP was measured in the stimulated cells using the cAMPSPA Direct Screening Assay System (Amersham) according to proceduresspecified by the manufacturer. As shown in FIG. 7, the guinea pig DPcell line exhibited a good cAMP response to PGD2 stimulation which wascomparable to the mouse DP receptor response. The EC50 values were 0.8nM and 0.5 nM for the guinea pig and mouse DP cell lines, respectivelyand the maximal response was comparable with both receptors. Incontrast, the parental HEK293-Gα16 cell line did not show an increase inintracellular cAMP in response to PGD2 stimulation.

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1. A recombinant polypeptide comprising the amino acid sequence of SEQID NO:2.
 2. The recombinant polypeptide of claim 1 further comprising adetectable label.
 3. The recombinant polypeptide of claim 2, wherein thedetectable label comprises an enzyme, a radio active isotope, or achemical which fluoresces.
 4. A recombinant polypeptide comprising anamino acid sequence at least 95% identical to the sequence of SEQ IDNO:2 and which binds a prostanoid.
 5. A recombinant polypeptidecomprising an amino acid sequence at least 99% identical to the sequenceof SEQ ID NO:2 and which binds a prostanoid.